Tricyclic Lactams for Use in the Protection of Normal Cells During Chemotherapy

ABSTRACT

This invention is in the area of tricyclic lactam compounds, compositions and methods of protecting healthy cells, and in particular hematopoietic stem and progenitor cells (HSPC) as well as renal cells, from damage associated with DNA damaging chemotherapeutic agents. In one aspect, protection of healthy cells is disclosed using compounds that act as cyclin-dependent kinase 4/6 (CDK 4/6) inhibitors when administered to subjects undergoing DNA damaging chemotherapeutic regimens for the treatment of proliferative disorders.

RELATED APPLICATIONS

This application claims the benefit of provisional U.S. Application No.61/980,883, filed Apr. 17, 2014, provisional U.S. Application No.61/980,895, filed Apr. 17, 2014, provisional U.S. Application No.61/980,918, filed Apr. 17, 2014, and provisional U.S. Application No.61/980,939, filed Apr. 17, 2014, which are hereby incorporated byreference for all purposes.

GOVERNMENT INTEREST

The U.S. Government has rights in this invention by virtue of supportunder Grant No. 5R44AI084284 awarded by the National Institute ofAllergy and Infectious Diseases.

FIELD OF THE INVENTION

This invention is in the area of tricyclic lactam compounds,compositions, and methods of protecting healthy cells, and in particularhematopoietic stem and progenitor cells (HSPC) as well as renal cells,from damage associated with DNA damaging chemotherapeutic agents.

BACKGROUND

Chemotherapy refers to the use of cytotoxic (typically DNA damaging)drugs to treat a range of proliferative disorders, including cancer,tumors, psoriasis, arthritis, lupus and multiple sclerosis, amongothers. Chemotherapeutic compounds tend to be non-specific and,particularly at high doses, toxic to normal, rapidly dividing cells.This often leads to a variety of side effects in patients undergoingchemotherapy.

Bone marrow suppression, a severe reduction of blood cell production inbone marrow, is one such side effect. It is characterized by bothmyelosuppression (anemia, neutropenia, agranulocytosis, andthrombocytopenia) and lymphopenia. Neutropenia is characterized by aselective decrease in the number of circulating neutrophils and anenhanced susceptibility to bacterial infections. Anemia, a reduction inthe number of red blood cells or erythrocytes, the quantity ofhemoglobin, or the volume of packed red blood cells (characterized by adetermination of the hematocrit) affects approximately 67% of cancerpatients undergoing chemotherapy in the United States. See BioWorldToday, page 4, Jul. 23, 2002. Thrombocytopenia is a reduction inplatelet number with increased susceptibility to bleeding. Lymphopeniais a common side-effect of chemotherapy characterized by a reduction inthe number of circulating lymphocytes (also called T- and B-cells).Lymphopenic patients are predisposed to a number of types of infections.

Myelosuppression continues to represent the major dose-limiting toxicityof cancer chemotherapy, resulting in considerable morbidity along withthe potential need to require a reduction in chemotherapy doseintensity, which may compromise disease control and survival.Considerable evidence from prospective and retrospective randomizedclinical trials clearly shows that chemotherapy-induced myelosuppressioncompromises long-term disease control and survival (Lyman, G. H.,Chemotherapy dose intensity and quality cancer care (Oncology (WillistonPark), 2006. 20(14 Suppl 9): p. 16-25)). Furthermore, treatment regimensfor, for example, lung, breast, and colorectal cancer recommended in theNational Comprehensive Cancer Network guidelines are increasinglyassociated with significant myelosuppression yet are increasinglyrecommended for treating early-stage disease as well as advanced-stageor metastatic disease (Smith, R. E., Trends in recommendations formyelosuppressive chemotherapy for the treatment of solid tumors. J NatlCompr Canc Netw, 2006. 4(7): p. 649-58). This trend toward moreintensive treatment of patients with cancer creates demand for measuresto minimize the risk of myelosuppression and complications whileoptimizing the relative dose-intensity.

In addition to bone marrow suppression, chemotherapeutic agents canadversely affect other healthy cells such as renal epithelial cells,resulting potentially in the development of acute kidney injury due tothe death of the tubular epithelia. Acute kidney injury can lead tochronic kidney disease, multi-organ failure, sepsis, and death.

One mechanism to minimize myelosuppression, nephrotoxicity, and otherchemotherapeutic cytotoxicities is to reduce the planned dose intensityof chemotherapies. Dose reductions or cycle delays, however, diminishthe effectiveness and ultimately compromise long-term disease controland survival.

Small molecules have been used to reduce some of the side effects ofcertain chemotherapeutic compounds. For example, leukovorin has beenused to mitigate the effects of methotrexate on bone marrow cells and ongastrointestinal mucosa cells. Amifostine has been used to reduce theincidence of neutropenia-related fever and mucositis in patientsreceiving alkylating or platinum-containing chemotherapeutics. Also,dexrazoxane has been used to provide cardioprotection from anthracyclineanti-cancer compounds. Unfortunately, there is concern that manychemoprotectants, such as dexrazoxane and amifostine, can decrease theefficacy of chemotherapy given concomitantly.

Additional chemoprotectant therapies, particularly with chemotherapyassociated anemia and neutropenia, include the use of growth factors.Hematopoietic growth factors are available on the market as recombinantproteins. These proteins include granulocyte colony stimulating factor(G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF)and their derivatives for the treatment of neutropenia, anderythropoietin (EPO) and its derivatives for the treatment of anemia.However, these recombinant proteins are expensive. Moreover, EPO hassignificant toxicity in cancer patients, leading to increasedthrombosis, relapse and death in several large randomized trials. G-CSFand GM-CSF may increase the late (>2 years post-therapy) risk ofsecondary bone marrow disorders such as leukemia and myelodysplasia.Consequently, their use is restricted and not readily available to allpatients in need. Further, while growth factors can hasten recovery ofsome blood cell lineages, no therapy exists to treat suppression ofplatelets, macrophages, T-cells or B-cells.

Hematopoietic stem cells give rise to progenitor cells which in turngive rise to all the differentiated components of blood as shown in FIG.1 (e.g., lymphocytes, erythrocytes, platelets, granulocytes, monocytes).HSPCs require the activity of Cyclin Dependent Kinase 4 (CDK4) andCyclin Dependent Kinase 6 (CDK6) for proliferation (see Roberts et al.Multiple Roles of Cyclin-Dependent Kinase 4/6 Inhibitors in CancerTherapy. JNCI 2012; 104(6):476-487). In healthy kidneys, the renalepithelium infrequently enters the cell cycle (about 1% of epithelialcells). After a renal insult, however, a robust increase in epithelialproliferation occurs (see Humphreys, B. D. et al. Intrinsic epithelialcells repair the kidney after injury. Cell Stem Cell 2, 284-91 (2008)).Importantly, following renal injury, surviving renal epithelial cellsreplicate to repair damage to the kidney tubular epithelium (seeHumphreys, B. D. et al. Repair of injured proximal tubule does notinvolve specialized progenitors. Proc Natl Acad Sci USA 108, 9226-31(2011); see also WO 2010132725 filed by Sharpless et al.).

A number of CDK4/6 inhibitors have been identified, including specificpyrido[2,3-d]pyrimidines, 2-anilinopyrimidines, diaryl ureas,benzoyl-2,4-diaminothiazoles, indolo[6,7-a]pyrrolo[3,4-c]carbazoles, andoxindoles (see P. S. Sharma, R. Sharma, R. Tyagi, Curr. Cancer DrugTargets 8 (2008) 53-75). WO 03/062236 identifies a series of2-(pyridin-2-ylamino-pyrido[2,3]pyrimidin-7-ones for the treatment of Rbpositive cancers that show selectivity for CDK4/6, including6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylammino)-8H-pyrido-[2,3-d]-pyrimidin-7-one(PD0332991). The clinical trial studies have reported rates of Grade 3/4neutropenia and leukopenia with the use of PD0332991, resulting in 71%of patients requiring a dose interruption and 35% requiring a dosereduction; and adverse events leading to 10% of the discontinuations(see Finn, Abstract S1-6, SABCS 2012).

VanderWel et al. describe an iodine-containingpyrido[2,3-d]pyrimidine-7-one (CKIA) as a potent and selective CDK4inhibitor (see VanderWel et al., J. Med. Chem. 48 (2005) 2371-2387).

WO 99/15500 filed by Glaxo Group Ltd discloses protein kinase andserine/threonine kinase inhibitors.

WO 2010/020675 filed by Novartis AG describes pyrrolopyrimidinecompounds as CDK inhibitors. WO 2011/101409 also filed by Novartisdescribes pyrrolopyrimidines with CDK 4/6 inhibitory activity.

WO 2005/052147 filed by Novartis and WO 2006/074985 filed by JanssenPharma disclose addition CDK4 inhibitors.

US 2007/0179118 filed by Barvian et al. teaches the use of CDK4inhibitors to treat inflammation.

U.S. Patent Publication 2011/0224227 to Sharpless et al. describes theuse of certain CDK4/6 inhibitors, such as PD0332991 and 2BrIC (see Zhu,et al., J. Med. Chem., 46 (11) 2027-2030 (2003); PCT/US2009/059281) toreduce or prevent the effects of cytotoxic compounds on HSPCs in asubject undergoing chemotherapeutic treatments. See also U.S. PatentPublication 2012/0100100.

U.S. Patent Publication 2011/0224221 to Sharpless et al. describes theuse of certain CDK4/6 inhibitors, such as PD0332991 and 2BrIC (see Zhu,et al., J. Med. Chem., 46 (11) 2027-2030 (2003); PCT/US2009/059281) toreduce or prevent the deleterious effects of ionizing radiation on HSPCsin a subject exposed to radiation. See also U.S. Patent Publication2012/0100100.

Stone, et al., Cancer Research 56, 3199-3202 (Jul. 1, 1996) describesreversible, p16-mediated cell cycle arrest as protection fromchemotherapy.

WO 2012/061156 filed by Tavares and assigned to G1 Therapeuticsdescribes CDK inhibitors (see also, U.S. Pat. Nos. 8,829,012, 8,822,683,8,598,186, 8,691,830, and 8,598,197, all assigned to G1 Therapeutics),describe CDK Inhibitors having the basic core structure:

WO 2013/148748 filed by Tavares and assigned to G1 Therapeuticsdescribes Lactam Kinase inhibitors having the basic core structures:

U.S. Patent Publication 2014/0275066 and 2014/0275067, assigned to G1Therapeutics, describes the use of CDK4/6 inhibitors such as2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-onefor the protection of healthy hematopoietic stem and progenitor cells ina subject receiving a DNA-damaging chemotherapeutic agent for thetreatment of a Rb-negative tumors.

U.S. Patent Publication 2014/0274896, assigned to G1 Therapeutics,describes the use of CDK4/6 inhibitors such as2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-onefor the protection of healthy hematopoietic stem and progenitor cells ina subject exposed to ionizing radiation.

U.S. Patent Publication 2014/0271466, assigned to G1 Therapeutics,describes the use of CDK4/6 inhibitors such as2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-onefor use as an antineoplastic for the treatment of a Rb-positiveproliferative disorders.

U.S. Patent Publication 2014/0271460, assigned to G1 Therapeutics,describes the use of CDK4/6 inhibitors such as2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-onefor use an antineoplastic for the treatment of a T- or B-cell disorder,for example a leukemia.

WO 2014/144326 assigned to G1 Therapeutics describes the use ofpyrrolopyrimidine compounds with CDK4/6 inhibitory activity, to reduceor prevent the effects of cytotoxic compounds on HSPCs in a subjectundergoing chemotherapeutic treatments.

Accordingly, it is an object of the present invention to provide newcompounds, compositions and methods to treat patients duringchemotherapy.

SUMMARY OF THE INVENTION

Methods and tricyclic lactam compounds are provided to minimize theeffect of chemotherapeutic agent toxicity on CDK4/6 replicationdependent healthy cells, such as hematopoietic stem cells andhematopoietic progenitor cells (together referred to as HSPCs), and/orrenal epithelial cells, in subjects, typically humans, that will be, arebeing, or have been exposed to the chemotherapeutic agent (typically aDNA-damaging agent).

The current invention provides for compounds of Formula I, II, III, IV,V, or VI, as described herein, a pharmaceutically acceptablecomposition, salt, isotopic analog, or prodrug thereof. In addition, thecurrent invention, provides for the compounds listed in Table 1.Specifically, the invention further includes administering an effectiveamount of a selected compound of Formula I, II, III, IV, V, or VI, asdescribed herein, a pharmaceutically acceptable composition, salt,isotopic analog, or prodrug thereof, which provides a G1-arrest ofhealthy cells, for example HSPCs and/or renal epithelial cells, in asubject prior to, during, or following the subject's exposure to achemotherapeutic agent, such as a DNA-damaging chemotherapeutic agent:

In one non-limiting example, a compound can be selected from thecompounds of Table 1 below, or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof.

The compounds described herein can be administered to the subject priorto treatment with a chemotherapeutic agent, during treatment with achemotherapeutic agent, after exposure to a chemotherapeutic agent, or acombination thereof. The compounds described herein are typicallyadministered in a manner that allows the drug facile access to the bloodstream, for example via intravenous injection or sublingual,intraaortal, or other efficient blood-stream accessing route; however,oral, topical, transdermal, intranasal, intramuscular, or by inhalationsuch as by a solution, suspension, or emulsion, or other desiredadministrative routes can be used. In one embodiment, the compound isadministered to the subject less than about 24 hours, 20 hours, 16hours, 12 hours, 8 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1 hour,½ hour or less prior to treatment with the chemotherapeutic agent.Typically, the active compound described herein is administered to thesubject prior to treatment with the chemotherapeutic agent such that thecompound reaches peak serum levels before or during treatment with thechemotherapeutic agent. In one embodiment, the active compound isadministered concomitantly, or closely thereto, with thechemotherapeutic agent exposure. If desired, the active compound can beadministered multiple times during the chemotherapeutic agent treatmentto maximize inhibition, especially when the chemotherapeutic drug isadministered over a long period or has a long half-life. The activecompound described herein can be administered following exposure to thechemotherapeutic agent if desired to mitigate healthy cell damageassociated with chemotherapeutic agent exposure. In certain embodiments,the active compound is administered up to about ½ hour, up to about 1hour, up to about 2 hours, up to about 4 hours, up to about 8 hours, upto about 10 hours, up to about 12 hours, up to about 14 hours, up toabout 16 hours, up to about 20 hours, or up to about 24 hours or greaterfollowing the chemotherapeutic agent exposure. In a particularembodiment, the active compound is administered up to between about 12hours and 20 hours following exposure to the chemotherapeutic agent. Inone embodiment, the tricyclic lactam is administered one or more timesfollowing exposure to chemotherapy.

In one embodiment, the tricyclic lactams described herein inhibit CyclinDependent Kinase 4 (CDK4) and/or Cyclin Dependent Kinase 6 (CDK6). Inone embodiment, the tricyclic lactams described herein may show a markedselectivity for the inhibition of CDK4 and/or CDK6 in comparison toother CDKs, for example CDK2. For example, a tricyclic lactam describedin the present invention may provide for a dose-dependent G1-arrestingeffect on a subject's CDK4/6-replication dependent healthy cells, forexample HSPCs or renal epithelial cells, and the methods provided forherein are sufficient to afford chemoprotection to targetedCDK4/6-replication dependent healthy cells during chemotherapeutic agentexposure, for example, during the time period that a DNA-damagingchemotherapeutic agent is capable of DNA-damaging effects onCDK4/6-replication dependent healthy cells in the subject. In oneembodiment, the use of a tricyclic lactam as described herein allows forthe synchronous and rapid reentry into the cell-cycle by these cellsshortly after the chemotherapeutic agent dissipates due to atime-limited CDK4/6 inhibitory effect.

In one embodiment, the use of the compounds or methods described hereinis combined with the use of hematopoietic growth factors including, butnot limited to, granulocyte colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF),thrombopoietin, interleukin (IL)-12, steel factor, and erythropoietin(EPO), or their derivatives. In one embodiment, the tricyclic lactam isadministered prior to administration of the hematopoietic growth factor.In one embodiment, the hematopoietic growth factor administration istimed so that the tricyclic lactam's effect on HSPCs has dissipated.

In one aspect, the use of a tricyclic lactam described herein allows fora chemo-protective regimen for use during standard chemotherapeuticdosing schedules or regimens common in many anti-cancer treatments. Forexample, the tricyclic lactam can be administered so thatCDK4/6-replication dependent healthy cells are G1 arrested duringchemotherapeutic agent exposure wherein, due to the dissipation of theG1-arresting effect of the compounds, a significant number of healthycells reenter the cell-cycle and are capable of replicating shortlyafter chemotherapeutic agent exposure, for example, within less thanabout 24, 30, 40, or 48 hours, and continue to replicate untiladministration of the tricyclic lactam in anticipation of the nextchemotherapeutic treatment. In one embodiment, the tricyclic lactam isadministered to allow for the cycling of the CDK4/6-replicationdependent healthy cells between G1-arrest and reentry into thecell-cycle to accommodate a repeated-dosing chemotherapeutic treatmentregimen, for example including but not limited to a treatment regimenwherein the chemotherapeutic agent is administered: on day 1-3 every 21days; on days 1-3 every 28 days; on day 1 every 3 weeks; on day 1, day8, and day 15 every 28 days, on day 1 and day 8 every 28 days; on days 1and 8 every 21 days; on days 1-5 every 21 days; 1 day a week for 6-8weeks; on days 1, 22, and 43; days 1 and 2 weekly; days 1-4 and 22-25;1-4; 22-25, and 43-46; and similar type-regimens, wherein theCDK4/6-replication dependent cells are G1 arrested duringchemotherapeutic agent exposure and a significant portion of the cellsreenter the cell-cycle between chemotherapeutic agent exposure. In oneembodiment, the tricyclic lactam can be administered so that thesubject's CDK4/6-replication dependent cells are G1-arrested duringdaily chemotherapeutic agent exposure, for example a contiguousmulti-day chemotherapeutic regimen, but a significant portion ofCDK4/6-replication dependent cells reenter the cell-cycle and replicatebetween daily treatment. In one embodiment, the tricyclic lactam can beadministered so that the subject's CDK4/6-replication dependent cellsare G1-arrested during chemotherapeutic agent exposure, for example acontiguous multi-day regimen, but a significant portion of healthy cellsreenter the cell-cycle and replicate during the off periods before thenext chemotherapeutic agent exposure. In one embodiment, the tricycliclactam is administered so that a subject's CDK4/6-replication dependentcells' G1-arrest is provided during a daily chemotherapeutic agenttreatment regimen, for example, a contiguous multi-day treatmentregimen, and the arrested cells are capable of reentering the cell-cycleshortly after the multi-day regimen ends. In one embodiment, the canceris small cell lung cancer and the tricyclic lactam is administered ondays 1, 2, and 3 during a 21-day treatment cycle wherein theadministered DNA damaging agent is selected from the group consisting ofcarboplatin, cisplatin, and etoposide, or a combination thereof.

The subject treated according to the present invention may be undergoingtherapeutic chemotherapy for the treatment of a proliferative disorderor disease such as cancer. The cancer can be characterized by one or acombination of increased activity of cyclin-dependent kinase 1 (CDK1),increased activity of cyclin-dependent kinase 2 (CDK2), loss,deficiency, or absence of retinoblastoma tumor suppressor protein(Rb)(Rb-null), high levels of MYC expression, increased cyclin E1, E2,and increased cyclin A. The cancer may be characterized by reducedexpression of the retinoblastoma tumor suppressor protein or aretinoblastoma family member protein or proteins (such as, but notlimited to p107 and p130). In one embodiment, the subject is undergoingchemotherapeutic treatment for the treatment of an Rb-null orRb-deficient cancer, including but not limited to small cell lungcancer, triple-negative breast cancer, HPV-positive head and neckcancer, retinoblastoma, Rb-negative bladder cancer, Rb negative prostatecancer, osteosarcoma, or cervical cancer. In one embodiment, the canceris a CDK4/6-independent cancer. Administration of the tricyclic lactamcompound may allow for a higher dose of a chemotherapeutic agent to beused to treat the disease than the standard dose that would be safelyused in the absence of administration of the tricyclic lactam compound.

The host or subject, including a human, may be undergoingchemotherapeutic treatment of a non-malignant proliferative disorder, orother abnormal cellular proliferation, such as a benign tumor, multiplesclerosis, lupus, or arthritis.

The protected HSPCs include hematopoietic stem cells, such as long termhematopoietic stem cells (LT-HSCs) and short term hematopoietic stemcells (ST-HSCs), and hematopoietic progenitor cells, includingmultipotent progenitors (MPPs), common myeloid progenitors (CMPs),common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors(GMPs) and megakaryocyte-erythroid progenitors (MEPs). Administration ofthe tricyclic lactam compound may provide temporary, transientpharmacologic quiescence of hematopoietic stem and/or hematopoieticprogenitor cells in the subject.

Administration of a tricyclic lactam as described herein can result inreduced anemia, reduced lymphopenia, reduced thrombocytopenia, orreduced neutropenia compared to that typically expected after, commonafter, or associated with treatment with chemotherapeutic agents in theabsence of administration of the tricyclic lactam.

In an alternative aspect, a tricyclic lactam described herein can alsobe used for its anti-cancer, anti-tumor, or anti-proliferative effect incombination with a chemotherapeutic agent to treat an Rb-negative canceror other Rb-negative abnormal proliferation. In one embodiment, atricyclic lactam described herein provides an additive effect to orsynergistic effect with the anti-cancer or anti-proliferative activityof the chemotherapeutic. Chemotherapeutics that can be combined with thetricyclic lactams described herein are any chemotherapeutics effectiveor useful to treat RB-null cancers or abnormal cellular proliferation.In one particular embodiment, the use of a compound described herein iscombined in a therapeutic regime with at least one otherchemotherapeutic agent, and can be one that does, or in certainembodiments does not, rely on proliferation or advancement through thecell-cycle for anti-proliferative activity. Such agent may include, butis not limited to, tamoxifen, midazolam, letrozole, bortezomib,anastrozole, goserelin, an mTOR inhibitor, a PI3 kinase inhibitors, dualmTOR-PI3K inhibitors, MEK inhibitors, RAS inhibitors, ALK inhibitors,HSP inhibitors (for example, HSP70 and HSP 90 inhibitors, or acombination thereof), BCL-2 inhibitors, apopototic inducing compounds,AKT inhibitors, PD-1 inhibitors, or FLT-3 inhibitors, or combinationsthereof. Examples of mTOR inhibitors include but are not limited torapamycin and its analogs, everolimus (Afinitor), temsirolimus,ridaforolimus, sirolimus, and deforolimus. Examples of P13 kinaseinhibitors include but are not limited to Wortmannin, demethoxyviridin,perifosine, idelalisib, PX-866, IPI-145 (Infinity), BAY 80-6946, BEZ235,RP6503, TGR 1202 (RP5264), MLN1117 (INK1117), Pictilisib, Buparlisib,SAR245408 (XL147), SAR245409 (XL765), Palomid 529, ZSTK474, PWT33597,RP6530, CUDC-907, and AEZS-136. Examples of MEK inhibitors include butare not limited to Tametinib, Selumetinib, MEK162, GDC-0973 (XL518), andPD0325901. Examples of RAS inhibitors include but are not limited toReolysin and siG12D LODER. Examples of ALK inhibitors include but arenot limited to Crizotinib, AP26113, and LDK378. HSP inhibitors includebut are not limited to Geldanamycin or17-N-Allylamino-17-demethoxygeldanamycin (17AAG), and Radicicol. Thetricyclic lactam combined with the chemotherapeutic is selected from thegroup consisting of a compound or composition comprising Formula I,Formula II, Formula III, Formula IV, Formula V, and Formula VI describedabove, or a pharmaceutically acceptable composition, salt, isotopicanalog or prodrug thereof. In one embodiment, the compound is selectedfrom the compounds provided for in Table 1, or a pharmaceuticallyacceptable composition, salt, isotopic analog or prodrug thereof.

In certain embodiments, a compound described herein is administered tothe subject prior to treatment with another chemotherapeutic agent,during treatment with another chemotherapeutic agent, afteradministration of another chemotherapeutic agent, or a combinationthereof. In one embodiment, the tricyclic lactam is selected from acompound described in Table 1.

In some embodiments, the subject or host is a mammal, including a human.

In summary, the present invention includes at least the followingfeatures:

A. Compounds of Formula I, II, III, IV, V, and VI as described herein,and pharmaceutically acceptable compositions, salts, isotopic analogs,or prodrugs thereof, for use in the chemoprotection ofCDK4/6-replication dependent healthy cells, for example HSPCs and/orrenal epithelial cells, during a chemotherapeutic agent exposure. In oneembodiment, the compound is selected from the compounds described inTable 1 or a pharmaceutically acceptable composition, salt, isotopicanalog or prodrug thereof.

B. Compounds of Formula I, II, III, IV, V, and VI as described herein,and pharmaceutically acceptable compositions, salts, isotopic analogs,and prodrugs thereof, for use in the chemoprotection ofCDK4/6-replication dependent healthy cells, for example HSPCs and/orrenal epithelial cells, during a chemotherapeutic regimen for thetreatment of a proliferative disorder. In one embodiment, the compoundis selected from the compounds described in Table 1 or apharmaceutically acceptable composition, salt, isotopic analog orprodrug thereof.

C. Compounds of Formula I, II, III, IV, V, and VI as described herein,and pharmaceutically acceptable compositions, salts, isotopic analogs,and prodrugs thereof, for use in the chemoprotection ofCDK4/6-replication dependent healthy cells, for example HSPCs and/orrenal epithelial cells, during a chemotherapeutic regimen for thetreatment of a cancer. In one embodiment, the compound is selected fromthe compounds described in Table 1, or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof.

D. Compounds of Formula I, II, III, IV, V, and VI as described herein,and pharmaceutically acceptable compositions, salts, isotopic analogs,and prodrugs thereof, for use in combination with hematopoietic growthfactors in a subject that will be, is being, or has been exposed tochemotherapeutic agents. In one embodiment, the compound is selectedfrom the compounds described in Table 1 or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof.

E. Use of compounds of Formula I, II, III, IV, V, and VI as describedherein, and pharmaceutically acceptable compositions, salts, isotopicanalogs, and prodrugs thereof, in the manufacture of a medicament foruse in the chemoprotection of CDK4/6-replication dependent healthycells, for example HSPCs and/or renal epithelial cells. In oneembodiment, the compound is selected from the compounds described inTable 1 or a pharmaceutically acceptable composition, salt, isotopicanalog or prodrug thereof.

F. Use of compounds of Formula I, II, III, IV, V, and VI as describedherein, and pharmaceutically acceptable compositions, salts, isotopicanalogs, and prodrugs thereof, in the manufacture of a medicament foruse in the mitigation of DNA damage of CDK4/6-replication dependenthealthy cells, for example HSPCs and/or renal epithelial cells, thathave been exposed to chemotherapeutic agent exposure. In one embodiment,the compound is selected from the compounds described in Table 1 or apharmaceutically acceptable composition, salt, isotopic analog orprodrug thereof.

G. A pharmaceutical formulation comprising an effective subject-treatingamount of compounds of Formula I, II, III, IV, V, and VI as describedherein, or pharmaceutically acceptable compositions, salts, and prodrugsthereof for use in chemoprotection of healthy cells. In one embodiment,the compound is selected from the compounds described in Table 1 or apharmaceutically acceptable composition, salt, isotopic analog orprodrug thereof.

H. A processes for the preparation of therapeutic products that containan effective amount of compounds of Formula I, II, III, IV, V, and VI asdescribed herein. In one embodiment, the compound is selected from thecompounds described in Table 1 or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof.

I. A method for manufacturing a medicament of Formula I, II, III, IV, V,and VI intended for therapeutic use in the chemoprotection ofCDK4/6-replication dependent healthy cells, for example HSPCs and/orrenal epithelial cells. In one embodiment, the medicament is selectedfrom the compounds described in Table 1 or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof.

J. A method for manufacturing a medicament of Formula I, II, III, IV, V,and VI intended for therapeutic use in the mitigation of DNA damage ofCDK4/6-replication dependent healthy cells, for example HSPCs and/orrenal epithelial cells, that have been exposed to chemotherapeuticagents. In one embodiment, the medicament is selected from the compoundsdescribed in Table 1 or a pharmaceutically acceptable composition, salt,isotopic analog or prodrug thereof.

K. A method of inhibiting the growth of an Rb-negative cancer orproliferative condition by administering a compound of Formula I, II,III, IV, V, or VI, or pharmaceutically acceptable composition, salt,isotopic analog or prodrug thereof; in combination with achemotherapeutic to provide an additive to or synergistic effect with achemotherapeutic. In one embodiment, the compound is selected from thecompounds described in Table 1 or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof. In oneembodiment, the tricyclic lactam is combined with a chemotherapeuticselected from the group consisting of MEK inhibitors, PI3 kinase deltainhibitors, BCL-2 inhibitors, AKT inhibitors, apoptotic inducingcompounds, AKT inhibitors, PD-1 inhibitors, FLT-3 inhibitors, HSP90inhibitors, or mTOR inhibitors, or combinations thereof.

L. Compounds of Formula I, II, III, IV, V, and VI as described herein,and pharmaceutically acceptable compositions, salts, isotopic analogs,or prodrugs thereof. In one embodiment, the compound is selected fromthe compounds described in Table 1 or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of hematopoiesis showing the hierarchicalproliferation of healthy hematopoietic stem cells (HSC) and healthyhematopoietic progenitor cells with increasing differentiation uponproliferation.

FIGS. 2-4 illustrate several exemplary embodiments of R² of thecompounds of the invention.

FIGS. 5A-5C, 6A-6D, 7A-7C, 8A-8B and 9A-9F illustrate exemplaryembodiments of the core structure of the compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Tricyclic lactam compounds, methods, and compositions are provided tominimize the effect of chemotherapeutic agent toxicity on CDK4/6replication dependent healthy cells, such as hematopoietic stem cellsand/or hematopoietic progenitor cells (together referred to as HSPCs),and/or renal epithelial cells, in subjects, typically humans, that willbe, are being or have been exposed to the chemotherapeutic agent(typically a DNA-damaging agent).

DEFINITIONS

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. As used in the specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Definition of standard chemistryterms may be found in reference works, including Carey and Sundberg(2007) Advanced Organic Chemistry 5^(th) Ed. Vols. A and B, SpringerScience+Business Media LLC, New York. The practice of the presentinvention will employ, unless otherwise indicated, conventional methodsof synthetic organic chemistry, mass spectroscopy, preparative andanalytical methods of chromatography, protein chemistry, biochemistry,recombinant DNA techniques and pharmacology. Conventional methods oforganic chemistry include those included in March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, 6^(th) Edition, M. B.Smith and J. March, John Wiley & Sons, Inc., Hoboken, N.J., 2007.

The term “alkyl,” either alone or within other terms such as “haloalkyl”and “alkylamino,” embraces linear or branched radicals having one toabout twelve carbon atoms. “Lower alkyl” radicals have one to about sixcarbon atoms. Examples of such radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl,hexyl and the like. The term “alkylene” embraces bridging divalentlinear and branched alkyl radicals. Examples include methylene,ethylene, propylene, isopropylene and the like.

The term “alkenyl” embraces linear or branched radicals having at leastone carbon-carbon double bond of two to about twelve carbon atoms.“Lower alkenyl” radicals having two to about six carbon atoms. Examplesof alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyland 4-methylbutenyl. The terms “alkenyl” and “lower alkenyl,” embraceradicals having “cis” and “trans” orientations, or alternatively, “E”and “Z” orientations.

The term “alkynyl” denotes linear or branched radicals having at leastone carbon-carbon triple bond and having two to about twelve carbonatoms. “Lower alkynyl” radicals having two to about six carbon atoms.Examples of such radicals include propargyl, butynyl, and the like.

Alkyl, alkenyl, and alkynyl radicals may be optionally substituted withone or more functional groups such as halo, hydroxy, nitro, amino,cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.

The term “alkylamino” embraces “N-alkylamino” and “N,N-dialkylamino”where amino groups are independently substituted with one alkyl radicaland with two alkyl radicals, respectively. “Lower alkylamino” radicalshave one or two alkyl radicals of one to six carbon atoms attached to anitrogen atom. Suitable alkylamino radicals may be mono or dialkylaminosuch as N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylaminoand the like.

The term “halo” means halogens such as fluorine, chlorine, bromine oriodine atoms.

The term “haloalkyl” embraces radicals wherein any one or more of thealkyl carbon atoms is substituted with one or more halo as definedabove. Examples include monohaloalkyl, dihaloalkyl and polyhaloalkylradicals including perhaloalkyl. A monohaloalkyl radical, for oneexample, may have an iodo, bromo, chloro or fluoro atom within theradical. Dihalo and polyhaloalkyl radicals may have two or more of thesame halo atoms or a combination of different halo radicals. “Lowerhaloalkyl” embraces radicals having 1-6 carbon atoms. Examples ofhaloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Perfluoroalkyl” means an alkyl radical having allhydrogen atoms replaced with fluoro atoms. Examples includetrifluoromethyl and pentafluoroethyl.

The term “aryl”, alone or in combination, means a carbocyclic aromaticsystem containing one or two rings wherein such rings may be attachedtogether in a fused manner. The term “aryl” embraces aromatic radicalssuch as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl. Morepreferred aryl is phenyl. Said “aryl” group may have 1 or moresubstituents such as lower alkyl, hydroxyl, halo, haloalkyl, nitro,cyano, alkoxy, lower alkylamino, and the like. An aryl group may beoptionally substituted with one or more functional groups such as halo,hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocycloand the like.

The term “heterocyclyl” (or “heterocyclo”) embraces saturated, andpartially saturated heteroatom-containing ring radicals, where theheteroatoms may be selected from nitrogen, sulfur and oxygen.Heterocyclic rings comprise monocyclic 6-8 membered rings, as well as5-16 membered bicyclic ring systems (which can include bridged fused andspiro-fused bicyclic ring systems). It does not include rings containing—O—O—.—O—S— or —S—S— portions. Said “heterocyclyl” group may have 1 to 3substituents such as hydroxyl, Boc, halo, haloalkyl, cyano, lower alkyl,lower aralkyl, oxo, lower alkoxy, amino, lower alkylamino, and the like.

Examples of saturated heterocyclo groups include saturated 3- to6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms[e.g. pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl,piperazinyl]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.morpholinyl]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g.,thiazolidinyl]. Examples of partially saturated heterocyclyl radicalsinclude dihydrothienyl, dihydropyranyl, dihydrofuryl, dihydrothiazolyl,and the like.

Particular examples of partially saturated and saturated heterocyclogroups include pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl,pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl,thiazolidinyl, dihydrothienyl, 2,3-dihydro-benzo[1,4]dioxanyl,indolinyl, isoindolinyl, dihydrobenzothienyl, dihydrobenzofuryl,isochromanyl, chromanyl, 1,2-dihydroquinolyl,1,2,3,4-tetrahydro-isoquinolyl, 1,2,3,4-tetrahydro-quinolyl,2,3,4,4a,9,9a-hexahydro-1H-3-aza-fluorenyl,5,6,7-trihydro-1,2,4-triazolo[3,4-a]isoquinolyl,3,4-dihydro-2H-benzo[1,4]oxazinyl, benzo[1,4]dioxanyl,2,3-dihydro-1H-1λ-benzo[d]isothiazol-6-yl, dihydropyranyl, dihydrofuryland dihydrothiazolyl, and the like.

Heterocyclo groups also includes radicals where heterocyclic radicalsare fused/condensed with aryl radicals: unsaturated condensedheterocyclic group containing 1 to 5 nitrogen atoms, for example,indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl,indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g.,tetrazolo[1,5-b]pyridazinyl]; unsaturated condensed heterocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.benzoxazolyl, benzoxadiazolyl]; unsaturated condensed heterocyclic groupcontaining 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g.,benzothiazolyl, benzothiadiazolyl]; and saturated, partially unsaturatedand unsaturated condensed heterocyclic group containing 1 to 2 oxygen orsulfur atoms [e.g. benzofuryl, benzothienyl,2,3-dihydro-benzo[1,4]dioxinyl and dihydrobenzofuryl].

The term “heteroaryl” denotes aryl ring systems that contain one or moreheteroatoms selected from the group O, N and S, wherein the ringnitrogen and sulfur atom(s) are optionally oxidized, and nitrogenatom(s) are optionally quarternized. Examples include unsaturated 5 to 6membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, forexample, pyrrolyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl,4-pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl [e.g.,4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl]; unsaturated5- to 6-membered heteromonocyclic group containing an oxygen atom, forexample, pyranyl, 2-furyl, 3-furyl, etc.; unsaturated 5 to 6-memberedheteromonocyclic group containing a sulfur atom, for example, 2-thienyl,3-thienyl, etc.; unsaturated 5- to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example,oxazolyl, isoxazolyl, oxadiazolyl [e.g., 1,2,4-oxadiazolyl,1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl]; unsaturated 5 to 6-memberedheteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3nitrogen atoms, for example, thiazolyl, thiadiazolyl [e.g.,1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl].

The term “heteroarylalkyl” denotes alkyl radicals substituted with aheteroaryl group. Examples include pyridylmethyl and thienylethyl.

The term “sulfonyl”, whether used alone or linked to other terms such asalkylsulfonyl, denotes respectively divalent radicals —SO₂—.

The terms “carboxy” or “carboxyl”, whether used alone or with otherterms, such as “carboxyalkyl”, denotes —C(O)—OH.

The term “carbonyl”, whether used alone or with other terms, such as“aminocarbonyl”, denotes —C(O)—.

The term “aminocarbonyl” denotes an amide group of the Formula—C(O)—NH₂.

The terms “heterocycloalkyl” embrace heterocyclic-substituted alkylradicals. Examples include piperidylmethyl and morpholinylethyl.

The term “arylalkyl” embraces aryl-substituted alkyl radicals. Examplesinclude benzyl, diphenylmethyl and phenylethyl. The aryl in said aralkylmay be additionally substituted with halo, alkyl, alkoxy, halkoalkyl andhaloalkoxy.

The term “cycloalkyl” includes saturated carbocyclic groups of 3 to 10carbons. Lower cycloalkyl groups include C₃-C₆ rings. Examples includecyclopentyl, cyclopropyl, and cyclohexyl. Cycloalkyl groups may beoptionally substituted with one or more functional groups such as halo,hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocycloand the like.

The term “cycloalkylalkyl” embraces cycloalkyl-substituted alkylradicals. “Lower cycloalkylalkyl” radicals are cycloalkyl radicalsattached to alkyl radicals having one to six carbon atoms. Examples ofinclude cyclohexylmethyl. The cycloalkyl in said radicals may beadditionally substituted with halo, alkyl, alkoxy and hydroxy.

The term “cycloalkenyl” includes carbocyclic groups having one or morecarbon-carbon double bonds including “cycloalkyldienyl” compounds.Examples include cyclopentenyl, cyclopentadienyl, cyclohexenyl andcycloheptadienyl.

The term “comprising” is meant to be open ended, including the indicatedcomponent but not excluding other elements.

The term “oxo” as used herein contemplates an oxygen atom attached witha double bond.

The term “nitro” as used herein contemplates —NO₂.

The term “cyano” as used herein contemplates —CN.

As used herein, the term “prodrug” means a compound which whenadministered to a host in vivo is converted into the parent drug. Asused herein, the term “parent drug” means any of the presently describedchemical compounds that are useful to treat any of the disordersdescribed herein, or to control or improve the underlying cause orsymptoms associated with any physiological or pathological disorderdescribed herein in a host, typically a human. Prodrugs can be used toachieve any desired effect, including to enhance properties of theparent drug or to improve the pharmaceutic or pharmacokinetic propertiesof the parent. Prodrug strategies exist which provide choices inmodulating the conditions for in vivo generation of the parent drug, allof which are deemed included herein. Nonlimiting examples of prodrugstrategies include covalent attachment of removable groups, or removableportions of groups, for example, but not limited to acylation,phosphorylation, phosphonylation, phosphoramidate derivatives,amidation, reduction, oxidation, esterification, alkylation, othercarboxy derivatives, sulfoxy or sulfone derivatives, carbonylation oranhydride, among others.

Throughout the specification and claims, a given chemical formula orname shall encompass all optical and stereoisomers, as well as racemicmixtures where such isomers and mixtures exist, unless otherwise noted.

In some embodiments, a CDK4/6-replication dependent healthy cell is ahematopoietic stem progenitor cell. Hematopoietic stem and progenitorcells include, but are not limited to, long term hematopoietic stemcells (LT-HSCs), short term hematopoietic stem cells (ST-HSCs),multipotent progenitors (MPPs), common myeloid progenitors (CMPs),common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors(GMPs), and megakaryocyte-erythroid progenitors (MEPs). In someembodiments, the CDK4/6-replication dependent healthy cell may be a cellin a non-hematopoietic tissue, such as, but not limited to, the liver,kidney, pancreas, brain, lung, adrenals, intestine, gut, stomach, skin,auditory system, bone, bladder, ovaries, uterus, testicles, gallbladder,thyroid, heart, pancreatic islets, blood vessels, and the like. In someembodiments, the CDK4/6-replication dependent healthy cell is a renalcell, and in particular a renal epithelial cell, for example, a renalproximal tubule epithelial cells. In some embodiments, aCDK4/6-replication dependent healthy cell is a hematopoietic stemprogenitor cell. In some embodiments, the CDK4/6-replication dependenthealthy cell may be a cell in a non-hematopoietic tissue, such as, butnot limited to, the liver, kidney, pancreas, brain, lung, adrenals,intestine, gut, stomach, skin, auditory system, bone, bladder, ovaries,uterus, testicles, gallbladder, thyroid, heart, pancreatic islets, bloodvessels, and the like.

The term “selective CDK4/6 inhibitor” used in the context of thecompounds described herein includes compounds that inhibit CDK4activity, CDK6 activity, or both CDK4 and CDK6 activity at an IC₅₀ molarconcentration at least about 500, or 1000, or 1500, or 1800, or 2000, or5000, or 10,000 times less than the IC₅₀ molar concentration necessaryto inhibit to the same degree of CDK2 activity in a standardphosphorylation assay.

By “induces G1-arrest” is meant that the inhibitor compound induces aquiescent state in a substantial portion of a cell population at the G1phase of the cell cycle.

By “hematological deficiency” is meant reduced hematological celllineage counts or the insufficient production of blood cells (i.e.,myelodysplasia) and/or lymphocytes (i.e., lymphopenia, the reduction inthe number of circulating lymphocytes, such as B- and T-cells).Hematological deficiency can be observed, for example, asmyelosuppression in form of anemia, reduction in platelet count (i.e.,thrombocytopenia), reduction in white blood cell count (i.e.,leukopenia), or the reduction in granulocytes (e.g., neutropenia).

By “synchronous reentry into the cell cycle” is meant thatCDK4/6-replication dependent healthy cells, for example HSPCs, inG1-arrest due to the effect of a tricyclic lactam compound reenter thecell-cycle within relatively the same collective timeframe or atrelatively the same rate upon dissipation of the compound's effect.Comparatively, by “asynchronous reentry into the cell cycle” is meantthat the healthy cells, for example HSPCs, in G1 arrest reenter thecell-cycle within relatively different collective timeframes or atrelatively different rates upon dissipation of the compound's effectsuch as PD0332991.

By “off-cycle” or “drug holiday” is meant a time period during which thesubject is not administered or exposed to a chemotherapeutic. Forexample, in a treatment regime wherein the subject is administered thechemotherapeutic for 21 straight days and is not administered thechemotherapeutic for 7 days, and the regime is repeated a number oftimes, the 7 day period of non-administration is considered the“off-cycle” or “drug holiday.” Off-cycle and drug holiday may also referto an interruption in a treatment regime wherein the subject is notadministered the chemotherapeutic for a time due to a deleterious sideeffect, for example, myelosuppression.

The subject treated is typically a human subject, although it is to beunderstood the methods described herein are effective with respect toother animals, such as mammals and vertebrate species. Moreparticularly, the term subject can include animals used in assays suchas those used in preclinical testing including but not limited to mice,rats, monkeys, dogs, pigs and rabbits; as well as domesticated swine(pigs and hogs), ruminants, equine, poultry, felines, bovines, murines,canines, and the like.

By “substantial portion” or “significant portion” is meant at least 80%.In alternative embodiments, the portion may be at least 85%, 90% or 95%or greater.

In some embodiments, the term “CDK4/6-replication independent cancer”refers to a cancer that does not significantly require the activity ofCDK4/6 for replication. Cancers of such type are often, but not always,characterized by (e.g., that has cells that exhibit) an increased levelof CDK2 activity or by reduced expression of retinoblastoma tumorsuppressor protein or retinoblastoma family member protein(s), such as,but not limited to p107 and p130. The increased level of CDK2 activityor reduced or deficient expression of retinoblastoma tumor suppressorprotein or retinoblastoma family member protein(s) can be increased orreduced, for example, compared to normal cells. In some embodiments, theincreased level of CDK2 activity can be associated with (e.g., canresult from or be observed along with) MYC proto-oncogene amplificationor overexpression. In some embodiments, the increased level of CDK2activity can be associated with overexpression of Cyclin E1, Cyclin E2,or Cyclin A.

As used herein the term “chemotherapy” or “chemotherapeutic agent”refers to treatment with a cytostatic or cytotoxic agent (i.e., acompound) to reduce or eliminate the growth or proliferation ofundesirable cells, for example cancer cells. Thus, as used herein,“chemotherapy” or “chemotherapeutic agent” refers to a cytotoxic orcytostatic agent used to treat a proliferative disorder, for examplecancer. The cytotoxic effect of the agent can be, but is not required tobe, the result of one or more of nucleic acid intercalation or binding,DNA or RNA alkylation, inhibition of RNA or DNA synthesis, theinhibition of another nucleic acid-related activity (e.g., proteinsynthesis), or any other cytotoxic effect.

Thus, a “cytotoxic agent” can be any one or any combination of compoundsalso described as “antineoplastic” agents or “chemotherapeutic agents.”Such compounds include, but are not limited to, DNA damaging compoundsand other chemicals that can kill cells. “DNA damaging chemotherapeuticagents” include, but are not limited to, alkylating agents, DNAintercalators, protein synthesis inhibitors, inhibitors of DNA or RNAsynthesis, DNA base analogs, topoisomerase inhibitors, and telomeraseinhibitors or telomeric DNA binding compounds. For example, alkylatingagents include alkyl sulfonates, such as busulfan, improsulfan, andpiposulfan; aziridines, such as a benzodizepa, carboquone, meturedepa,and uredepa; ethylenimines and methylmelamines, such as altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, cyclophosphamide, estramustine,iphosphamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichine, phenesterine, prednimustine, trofosfamide, anduracil mustard; and nitroso ureas, such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, and ranimustine.

Antibiotics used in the treatment of cancer include dactinomycin,daunorubicin, doxorubicin, idarubicin, bleomycin sulfate, mytomycin,plicamycin, and streptozocin. Chemotherapeutic antimetabolites includemercaptopurine, thioguanine, cladribine, fludarabine phosphate,fluorouracil (5-FU), floxuridine, cytarabine, pentostatin, methotrexate,and azathioprine, acyclovir, adenine β-1-D-arabinoside, amethopterin,aminopterin, 2-aminopurine, aphidicolin, 8-azaguanine, azaserine,6-azauracil, 2′-azido-2′-deoxynucleosides, 5-bromodeoxycytidine,cytosine β-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides,5-fluorodeoxycytidine, 5-fluorodeoxyuridine, and hydroxyurea.

Chemotherapeutic protein synthesis inhibitors include abrin,aurintricarboxylic acid, chloramphenicol, colicin E3, cycloheximide,diphtheria toxin, edeine A, emetine, erythromycin, ethionine, fluoride,5-fluorotryptophan, fusidic acid, guanylyl methylene diphosphonate andguanylyl imidodiphosphate, kanamycin, kasugamycin, kirromycin, andO-methyl threonine. Additional protein synthesis inhibitors includemodeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin,ricin, shiga toxin, showdomycin, sparsomycin, spectinomycin,streptomycin, tetracycline, thiostrepton, and trimethoprim Inhibitors ofDNA synthesis, include alkylating agents such as dimethyl sulfate,mitomycin C, nitrogen and sulfur mustards; intercalating agents, such asacridine dyes, actinomycins, adriamycin, anthracenes, benzopyrene,ethidium bromide, propidium diiodide-intertwining; and other agents,such as distamycin and netropsin. Topoisomerase inhibitors, such ascoumermycin, nalidixic acid, novobiocin, and oxolinic acid; inhibitorsof cell division, including colcemide, colchicine, vinblastine, andvincristine; and RNA synthesis inhibitors including actinomycin D,α-amanitine and other fungal amatoxins, cordycepin (3′-deoxyadenosine),dichlororibofuranosyl benzimidazole, rifampicine, streptovaricin, andstreptolydigin also can be used as the DNA damaging compound.

Current chemotherapeutic agents whose toxic effects can be mitigated bythe presently disclosed tricyclic lactams include, but are not limitedto, adrimycin, 5-fluorouracil (5FU), 6-mercaptopurine, gemcitabine,melphalan, chlorambucil, mitomycin, irinotecan, mitoxantrone, etoposide,camptothecin, topotecan, irinotecan, exatecan, lurtotecan,actinomycin-D, mitomycin, cisplatin, hydrogen peroxide, carboplatin,procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, tamoxifen, taxol, transplatinum,vinblastine, vinblastin, carmustine, cytarabine, mechlorethamine,chlorambucil, streptozocin, lomustine, temozolomide, thiotepa,altretamine, oxaliplatin, campothecin, and methotrexate, and the like,and similar acting-type agents. In one embodiment, the DNA damagingchemotherapeutic agent is selected from the group consisting ofcisplatin, carboplatin, campothecin, doxorubicin, and etoposide.

In certain alternative embodiments, the tricyclic lactams describedherein are also used for an anti-cancer or anti-proliferative effect incombination with a chemotherapeutic to treat a CDK4/6 replicationindependent, such as an Rb-negative, cancer or proliferative disorder.The tricyclic lactams described herein may provide an additive orsynergistic effect to the chemotherapeutic, resulting in a greateranti-cancer effect than seen with the use of the chemotherapeutic alone.In one embodiment, the tricyclic lactams described herein can becombined with one or more of the chemotherapeutic compounds describedabove. In one embodiment, a tricyclic lactam described herein can becombined with a chemotherapeutic selected from, but not limited to, butnot limited to, tamoxifen, midazolam, letrozole, bortezomib,anastrozole, goserelin, an mTOR inhibitor, a PI3 kinase inhibitors, dualmTOR-PI3K inhibitors, MEK inhibitors, RAS inhibitors, ALK inhibitors,HSP inhibitors (for example, HSP70 and HSP 90 inhibitors, or acombination thereof), BCL-2 inhibitors, apopototic inducing compounds,AKT inhibitors, including but not limited to, MK-2206, GSK690693,Perifosine, (KRX-0401), GDC-0068, Triciribine, AZD5363, Honokiol,PF-04691502, and Miltefosine, PD-1 inhibitors including but not limitedto, Nivolumab, CT-011 (pidilizumab), MK-3475 (pembrolizumab), BMS936558,MPDL328OA (Roche), and AMP-514 or FLT-3 inhibitors, including but notlimited to, P406, Dovitinib, Quizartinib (AC220), Amuvatinib (MP-470),Tandutinib (MLN518), ENMD-2076, and KW-2449, or combinations thereof.Examples of mTOR inhibitors include but are not limited to rapamycin andits analogs, everolimus (Afinitor), temsirolimus, ridaforolimus,sirolimus, and deforolimus. Examples of P13 kinase inhibitors includebut are not limited to Wortmannin, demethoxyviridin, perifosine,idelalisib, PX-866, IPI-145 (Infinity), BAY 80-6946, BEZ235, RP6503, TGR1202 (RP5264), MLN1117 (INK1117), Pictilisib, Buparlisib, SAR245408(XL147), SAR245409 (XL765), Palomid 529, ZSTK474, PWT33597, RP6530,CUDC-907, and AEZS-136. Examples of MEK inhibitors include but are notlimited to Tametinib, Selumetinib, MEK162, GDC-0973 (XL518), andPD0325901. Examples of RAS inhibitors include but are not limited toReolysin and siGl2D LODER. Examples of ALK inhibitors include but arenot limited to Crizotinib, AP26113, and LDK378. HSP inhibitors includebut are not limited to Geldanamycin or17-N-Allylamino-17-demethoxygeldanamycin (17AAG), and Radicicol. In aspecific embodiment, a tricyclic lactam is combined with camptothecin,or a derivative thereof, such as topotecan, irinotecan, exatecan, orlurotecan, or a combination thereof. In one embodiment, the tricycliclactams combined with the chemotherapeutic is selected from the groupconsisting of a compound or composition comprising Formula I, FormulaII, Formula III, Formula IV, Formula V, and Formula VI described above,or a pharmaceutically acceptable composition, salt, isotopic analog orprodrug thereof. In one embodiment, the compound is selected from thecompounds provided for in Table 1, or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof.

In one embodiment, a tricyclic lactam described herein can be combinedwith a chemotherapeutic selected from, but are not limited to, Imatinibmesylate (Gleevac®), Dasatinib (Sprycel®), Nilotinib (Tasigna®),Bosutinib (Bosulif®), Trastuzumab (Herceptin®), Pertuzumab (Perjeta™),Lapatinib (Tykerb®), Gefitinib (Iressa®), Erlotinib (Tarceva®),Cetuximab (Erbitux®), Panitumumab (Vectibix®), Vandetanib (Caprelsa®),Vemurafenib (Zelboraf®), Vorinostat (Zolinza®), Romidepsin (Istodax®),Bexarotene (Tagretin®), Alitretinoin (Panretin®), Tretinoin (Vesanoid®),Carfilizomib (Kyprolis™), Pralatrexate (Folotyn®), Bevacizumab(Avastin®), Ziv-aflibercept (Zaltrap®), Sorafenib (Nexavar®), Sunitinib(Sutent®), Pazopanib (Votrient®), Regorafenib (Stivarga®), andCabozantinib (Cometriq™).

In one aspect of the present invention, a compound described herein canbe combined with at least one anti-inflammatory agent. Theanti-inflammatory agent can be a steroidal anti-inflammatory agent, anonsteroidal anti-inflammatory agent, or a combination thereof. In someembodiments, anti-inflammatory drugs include, but are not limited to,alclofenac, alclometasone dipropionate, algestone acetonide, alphaamylase, amcinafal, amcinafide, amfenac sodium, amiprilosehydrochloride, anakinra, anirolac, anitrazafen, apazone, balsalazidedisodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelains,broperamole, budesonide, carprofen, cicloprofen, cintazone, cliprofen,clobetasol propionate, clobetasone butyrate, clopirac, cloticasonepropionate, cormethasone acetate, cortodoxone, deflazacort, desonide,desoximetasone, dexamethasone dipropionate, diclofenac potassium,diclofenac sodium, diflorasone diacetate, diflumidone sodium,diflunisal, difluprednate, diftalone, dimethyl sulfoxide, drocinonide,endrysone, enlimomab, enolicam sodium, epirizole, etodolac, etofenamate,felbinac, fenamole, fenbufen, fenclofenac, fenclorac, fendosal,fenpipalone, fentiazac, flazalone, fluazacort, flufenamic acid,flumizole, flunisolide acetate, flunixin, flunixin meglumine, fluocortinbutyl, fluorometholone acetate, fluquazone, flurbiprofen, fluretofen,fluticasone propionate, furaprofen, furobufen, halcinonide, halobetasolpropionate, halopredone acetate, ibufenac, ibuprofen, ibuprofenaluminum, ibuprofen piconol, ilonidap, indomethacin, indomethacinsodium, indoprofen, indoxole, intrazole, isoflupredone acetate,isoxepac, isoxicam, ketoprofen, lofemizole hydrochloride, lomoxicam,loteprednol etabonate, meclofenamate sodium, meclofenamic acid,meclorisone dibutyrate, mefenamic acid, mesalamine, meseclazone,methylprednisolone suleptanate, morniflumate, nabumetone, naproxen,naproxen sodium, naproxol, nimazone, olsalazine sodium, orgotein,orpanoxin, oxaprozin, oxyphenbutazone, paranyline hydrochloride,pentosan polysulfate sodium, phenbutazone sodium glycerate, pirfenidone,piroxicam, piroxicam cinnamate, piroxicam olamine, pirprofen,prednazate, prifelone, prodolic acid, proquazone, proxazole, proxazolecitrate, rimexolone, romazarit, salcolex, salnacedin, salsalate,sanguinarium chloride, seclazone, sermetacin, sudoxicam, sulindac,suprofen, talmetacin, talniflumate, talosalate, tebufelone, tenidap,tenidap sodium, tenoxicam, tesicam, tesimide, tetrydamine, tiopinac,tixocortol pivalate, tolmetin, tolmetin sodium, triclonide,triflumidate, zidometacin, zomepirac sodium, aspirin (acetylsalicylicacid), salicylic acid, corticosteroids, glucocorticoids, tacrolimus,pimecorlimus, prodrugs thereof, co-drugs thereof, and combinationsthereof.

In one aspect of the present invention, a compound described herein canbe combined with at least one immunomodulatory agent. In one embodiment,the immunomodulatory agent is selected from the group consisting of aCTLA-4 inhibitor, PD-1 or anti-PD-1 agent, IFN-alpha, IFN-beta, and avaccine, for example, a cancer vaccine. In one embodiment, the PD-1agent is Keytruda® (pembrolizumab). In one embodiment, the PD-1 agent isOpdivo (nivolumab). In one embodiment, the CTLA-4 inhibitor is Yervoy®(ipilimumab).

By “long-term hematological toxicity” is meant hematological toxicityaffecting a subject for a period lasting more than one or more weeks,months, or years following administration of a chemotherapeutic agent.Long-term hematological toxicity can result in bone marrow disordersthat can cause the ineffective production of blood cells (i.e.,myelodysplasia) and/or lymphocytes (i.e., lymphopenia, the reduction inthe number of circulating lymphocytes, such as B- and T-cells).Hematological toxicity can be observed, for example, as anemia,reduction in platelet count (i.e., thrombocytopenia) or reduction inwhite blood cell count (i.e., neutropenia). In some cases,myelodysplasia can result in the development of leukemia. Long-termtoxicity related to chemotherapeutic agents can also damage otherself-renewing cells in a subject, in addition to hematological cells.Thus, long-term toxicity can also lead to graying and frailty.

Active Compounds

In one embodiment, the invention is directed to compounds or the use ofsuch compounds of Formula I, II, III, IV, or V:

or a pharmaceutically acceptable salt thereof;wherein:Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)— wherein z is2, 3 or 4;each X is independently CH or N;each X′ is independently CH or N;X″ is independently CH₂, S or NH, arranged such that the moiety is astable 5-membered ring; R, R⁸, and R¹¹ are independently H, C₁-C₃ alkylor haloalkyl, cycloalkyl or cycloalkyl containing one or moreheteroatoms selected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl,-(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valence, and wherein two R^(x) groups bound to thesame or adjacent atoms may optionally combine to form a ring;each R¹ is independently aryl, alkyl, cycloalkyl or haloalkyl, whereineach of said alkyl, cycloalkyl and haloalkyl groups optionally includesO or N heteroatoms in place of a carbon in the chain and two R¹'s onadjacent ring atoms or on the same ring atom together with the ringatom(s) to which they are attached optionally form a 3-8-membered cycle;y is 0, 1, 2, 3 or 4;R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴;-(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valence, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring and whereinm is 0, 1 or 2 and n is 0, 1 or 2;R³ and R⁴ at each occurrence are independently:

-   -   (i) hydrogen or    -   (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,        cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl        any of which may be optionally independently substituted with        one or more R^(x) groups as allowed by valence, and wherein two        R^(x) groups bound to the same or adjacent atom may optionally        combine to form a ring; or R³ and R⁴ together with the nitrogen        atom to which they are attached may combine to form a        heterocyclo ring optionally independently substituted with one        or more R^(x) groups as allowed by valence, and wherein two        R^(x) groups bound to the same or adjacent atom may optionally        combine to form a ring;        R⁵ and R⁵* at each occurrence is:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which may be optionally independently        substituted with one or more R^(x) groups as allowed by valence;        R^(x) at each occurrence is independently, halo, cyano, nitro,        oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl,        -(alkylene)_(m)-OR⁵, -(alkylene)_(m)-O-alkylene-OR⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵,        -(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵,        -(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵,        -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵,        -(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴,        -(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(O)—OR⁵) -(alkylene)_(m)-N(R³)—C(S)—OR⁵,        or -(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein:    -   said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups        may be further independently substituted with one or more        -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵*,        -(alkylene)_(m)-S(O)_(n)—R⁵*, -(alkylene)_(m)-NR³*R⁴*,        -(alkylene)_(m)-C(O)—R⁵*, -(alkylene)_(m)-C(═S)R⁵*,        -(alkylene)_(m)-C(═O)OR⁵*, -(alkylene)_(m)-OC(═O)R⁵*,        -(alkylene)_(m)-C(S)—OR⁵*, -(alkylene)_(m)-C(O)—NR³*R⁴*,        -(alkylene)_(m)-C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,        -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,        -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,    -   n is 0, 1 or 2, and    -   m is 0, 1 or 2;        R³* and R⁴* at each occurrence are independently:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which may be optionally independently        substituted with one or more R^(x) groups as allowed by valence;        or R³* and R⁴* together with the nitrogen atom to which they are        attached may combine to form a heterocyclo ring optionally        independently substituted with one or more R^(x) groups as        allowed by valence; and        R⁶ is H or lower alkyl, -(alkylene)_(m)-heterocyclo,        -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴        any of which may be optionally independently substituted with        one or more R^(x) groups as allowed by valence, and wherein two        R^(x) groups bound to the same or adjacent atoms may optionally        combine to form a ring; and        R¹⁰ is 1 (i) NHR^(A), wherein R^(A) is unsubstituted or        substituted C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈        cycloalkyl or cycloalkyl containing one or more heteroatoms        selected from N, O, and S; TT is an unsubstituted or substituted        C₁-C₈ alkyl or C₃-C₈ cycloalkyl linker; and RR is a hydroxyl,        unsubstituted or substituted C₁-C₆ alkoxy, amino, unsubstituted        or substituted C₁-C₆ alkylamino, unsubstituted or substituted        di-C₁-C₆ alkylamino, unsubstituted or substituted C₆-C₁₀ aryl,        unsubstituted or substituted heteroaryl comprising one or two 5-        or 6-member rings and 1-4 heteroatoms selected from N, O and S,        unsubstituted or substituted C₃-C₁₀ carbocycle, or unsubstituted        or substituted heterocycle comprising one or two 5- or 6-member        rings and 1-4 heteroatoms selected from N, O and S; or (ii)        —C(O)—R¹² or —C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³        is R^(A);        when compounds comprise a double bond in the 6-membered ring        fused to the pyrimidine ring, two R⁸ groups are present and are        as defined above;        when compounds do not comprise a double bond in the 6-membered        ring fused to the pyrimidine ring, four R⁸ groups are present        and are as defined above;        or a pharmaceutically acceptable salt, prodrug or isotopic        variant, for example, partially or fully deuterated form        thereof.

In one embodiment, two R⁸ groups bonded to the same carbon can form anexocyclic double bond. In another embodiment, two R⁸ groups bonded tothe same carbon can form a carbonyl group.

In one embodiment, the invention is directed to compounds or the use ofsuch compounds of Formula VI:

wherein R, R¹, R², R³, R⁴, R⁵, R⁶, R^(x), Z, m, n, and y are as definedabove;each R¹⁴ is independently H, C₁-C₃ alkyl (including methyl) orhaloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatomsselected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl,-(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valence, and wherein two R^(x) groups bound to thesame or adjacent atoms may optionally combine to form a ring;or two R¹⁴ groups bonded to the same carbon can form an exocyclic doublebond;or two R¹⁴ groups bonded to the same carbon can form a carbonyl group;and when the compound of Formula VI has a double bond, as indicated bythe ( - - - - ), in the 6-membered ring fused to the pyrimidine ring,two R¹⁴ groups are present as allowed for in Formula VI above; orwhen the compound of Formula VI does not include a double bond, asindicated by the ( - - - - ), in the 6-membered ring fused to thepyrimidine ring, four R¹⁴ groups are present as allowed for in FormulaVI above;or a pharmaceutically acceptable salt, prodrug or isotopic variant, forexample, partially or fully deuterated form thereof.

In an alternative embodiment, the invention is directed to compounds orthe use of such compounds of Formula I, II, III, IV, or V:

or a pharmaceutically acceptable salt thereof;wherein:Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)— wherein z is2, 3 or 4;each X is independently CH or N;each X′ is independently CH or N;X″ is independently CH₂, S or NH, arranged such that the moiety is astable 5-membered ring;R, R⁸, and R¹¹ are independently H, C₁-C₃ alkyl (including methyl) orhaloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatomsselected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl,-(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich, other than heterocyclo, may be optionally independentlysubstituted with one or more R^(x) groups as allowed by valence, andwherein two R^(x) groups bound to the same or adjacent atoms mayoptionally combine to form a ring;each R¹ is independently aryl, alkyl, cycloalkyl or haloalkyl, whereineach of said alkyl, cycloalkyl and haloalkyl groups optionally includesO or N heteroatoms in place of a carbon in the chain and two R¹'s onadjacent ring atoms or on the same ring atom together with the ringatom(s) to which they are attached optionally form a 3-8-membered cycle;y is 0, 1, 2, 3 or 4;R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴;-(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich, other than heterocyclo, may be optionally independentlysubstituted with one or more R^(x) groups as allowed by valence, andwherein two R^(x) groups bound to the same or adjacent atom mayoptionally combine to form a ring and wherein m is 0, 1, or 2 and n is0, 1 or 2;wherein heterocyclo may be optionally independently substituted with 1to 3 R^(x) groups as allowed by valence, and wherein two R^(x) groupsbound to the same or adjacent atom may optionally combine to form aring;R³ and R⁴ at each occurrence are independently:

-   -   (i) hydrogen or    -   (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,        cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl        any of which, other than heterocyclo, may be optionally        independently substituted with one or more R^(x) groups as        allowed by valence, and wherein two R^(x) groups bound to the        same or adjacent atom may optionally combine to form a ring; or        R³ and R⁴ together with the nitrogen atom to which they are        attached may combine to form a heterocyclo ring optionally        independently substituted with one or more R^(x) groups as        allowed by valence, and wherein two R^(x) groups bound to the        same or adjacent atom may optionally combine to form a ring;        R⁵ and R⁵* at each occurrence is:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which, other than heterocyclo, may be        optionally independently substituted with one or more R^(x)        groups as allowed by valence;        R^(x) at each occurrence is independently, halo, cyano, nitro,        oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl,        -(alkylene)_(m)-OR⁵, -(alkylene)_(m)-O-alkylene-OR⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵,        -(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵,        -(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵,        -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵,        -(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴,        -(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(O)—OR⁵, -(alkylene)_(m)-N(R³)—C(S)—OR⁵,        or -(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein:    -   said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups,        any of which, other than heterocyclo, may be further        independently substituted with one or more -(alkylene)_(m)-CN,        -(alkylene)_(m)-OR⁵*, -(alkylene)_(m)-S(O)_(n)—R⁵*,        -(alkylene)_(m)-NR³*R⁴*, -(alkylene)_(m)-C(O)—R⁵*,        -(alkylene)_(m)-C(═S)R⁵*, -(alkylene)_(m)-C(═O)OR⁵*,        -(alkylene)_(m)-OC(═O)R⁵*, -(alkylene)_(m)-C(S)—OR⁵*,        -(alkylene)_(m)-C(O)—NR³*R⁴*, -(alkylene)_(m)-C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,        -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,        -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*, and    -   wherein heterocycle may be further independently substituted        with one to three substitutions selected from    -   -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵*,        -(alkylene)_(m)-S(O)_(n)—R⁵*, -(alkylene)_(m)-NR³*R⁴*,        -(alkylene)_(m)-C(O)—R⁵*, -(alkylene)_(m)-C(═S)R⁵*,        -(alkylene)_(m)-C(═O)OR⁵*, -(alkylene)_(m)-OC(═O)R⁵*,        -(alkylene)_(m)-C(S)—OR⁵*, -(alkylene)_(m)-C(O)—NR³*R⁴*,        -(alkylene)_(m)-C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,        -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,        -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*;    -   n is 0, 1 or 2, and    -   m is 0, 1; or 2 and        R³* and R⁴* at each occurrence are independently:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which, other than heterocyclo, may be        optionally independently substituted with one or more R^(x)        groups as allowed by valence; or R³* and R⁴* together with the        nitrogen atom to which they are attached may combine to form a        heterocyclo ring optionally independently substituted with one        or more R^(x) groups as allowed by valence;        R⁶ is H, absent, or lower alkyl, -(alkylene)_(m)-heterocyclo,        -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴        any of which, other than heterocyclo, may be optionally        independently substituted with one or more R^(x) groups as        allowed by valence, and wherein two R^(x) groups bound to the        same or adjacent atoms may optionally combine to form a ring;        and        R¹⁰ is 1 (i) NHR^(A), wherein R^(A) is unsubstituted or        substituted C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈        cycloalkyl or cycloalkyl containing one or more heteroatoms        selected from N, O, and S; TT is an unsubstituted or substituted        C₁-C₈ alkyl or C₃-C₈ cycloalkyl linker; and RR is a hydroxyl,        unsubstituted or substituted C₁-C₆ alkoxy, amino, unsubstituted        or substituted C₁-C₆ alkylamino, unsubstituted or substituted        di-C₁-C₆ alkylamino, unsubstituted or substituted C₆-C₁₀ aryl,        unsubstituted or substituted heteroaryl comprising one or two 5-        or 6-member rings and 1-4 heteroatoms selected from N, O and S,        unsubstituted or substituted C₃-C₁₀ carbocycle, or unsubstituted        or substituted heterocycle comprising one or two 5- or 6-member        rings and 1-4 heteroatoms selected from N, O and S; or (ii)        —C(O)—R¹² or —C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³        is R^(A);        when the compound of Formula I, II, III, IV, or V has a double        bond, as indicated by the ( - - - - ), in the 6-membered ring        fused to the pyrimidine ring, two R⁸ groups are present as        allowed for in Formula I, II, III, IV, or V above; or        when the compound of Formula I, II, III, IV, or V does not        include a double bond, as indicated by the ( - - - - ), in the        6-membered ring fused to the pyrimidine ring, four R⁸ groups are        present as allowed for in Formula I, II, III, IV, or V above;

-   wherein each heteroaryl is an aryl ring system that contains one or    more heteroatoms selected from the group O, N and S, wherein the    ring nitrogen and sulfur atom(s) are optionally oxidized, and    nitrogen atom(s) are optionally quarternized;

-   wherein each aryl is a carbocyclic aromatic system containing one or    two rings, wherein such rings may be attached together in a fused    manner, and wherein each aryl may have 1 or more R^(x) substituents;

-   wherein each heterocyclo is a saturated or partially saturated    heteroatom-containing ring radical, where the heteroatoms may be    selected from nitrogen, sulfur and oxygen, wherein each heterocyclo    is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring    system, and wherein each heterocyclo may have 1 to 3 R^(x)    substituents;    or a pharmaceutically acceptable salt, prodrug or isotopic variant,    for example, partially or fully deuterated form thereof.

In an alternative embodiment, the term “aryl” means a carbocyclicaromatic system containing one or two rings wherein such rings may beattached together in a fused manner, which may have 1 or moresubstituents selected from lower alkyl, hydroxyl, halo, haloalkyl,nitro, cyano, alkoxy and lower alkylamino.

In an alternative embodiment, the term “heterocyclyl” or “heterocyclo”means a saturated or partially saturated heteroatom-containing ringradical, where the heteroatoms may be selected from nitrogen, sulfur andoxygen, which may have 1 to 3 substituents selected from hydroxyl, Boc,halo, haloalkyl, cyano, lower alkyl, lower aralkyl, oxo, lower alkoxy,amino and lower alkylamino, wherein the heterocyclic ring is amonocyclic 6-8 membered rings, or a 5-16 membered bicyclic ring systemswhich can include bridged fused and spiro-fused bicyclic ring systems,and which does not include rings containing —O—O—.—O—S— or —S—S—portion.

In an alternative embodiment, the term “heteroaryl” means an aryl ringsystem that contains one or more heteroatoms selected from the group O,N and S, wherein the ring nitrogen and sulfur atom(s) are optionallyoxidized, and nitrogen atom(s) are optionally quarternized.

In one embodiment, two R⁸ groups bonded to the same carbon can form anexocyclic double bond. In another embodiment, two R⁸ groups bonded tothe same carbon can form a carbonyl group.

In an alternative embodiment, the invention is directed to compounds orthe use of such compounds of Formula VI:

wherein R, R¹, R², R³, R⁴, R⁵, R⁶, R^(x), Z, m, n, and y are as definedabove;each R¹⁴ is independently H, C₁-C₃ alkyl (including methyl) orhaloalkyl, cycloalkyl or cycloalkyl containing one or more heteroatomsselected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl,-(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich, other than heterocyclo, may be optionally independentlysubstituted with one or more R^(x) groups as allowed by valence, andwherein two R^(x) groups bound to the same or adjacent atoms mayoptionally combine to form a ring;or two R¹⁴ groups bonded to the same carbon can form an exocyclic doublebond;or two R¹⁴ groups bonded to the same carbon can form a carbonyl group;and when the compound of Formula VI has a double bond, as indicated bythe ( - - - - ), in the 6-membered ring fused to the pyrimidine ring,two R¹⁴ groups are present as allowed for in Formula VI above; orwhen the compound of Formula VI does not include a double bond, asindicated by the ( - - - - ), in the 6-membered ring fused to thepyrimidine ring, four R¹⁴ groups are present as allowed for in FormulaVI above;

-   wherein each heteroaryl is an aryl ring system that contains one or    more heteroatoms selected from the group O, N and S, wherein the    ring nitrogen and sulfur atom(s) are optionally oxidized, and    nitrogen atom(s) are optionally quarternized;-   wherein each aryl is a carbocyclic aromatic system containing one or    two rings, wherein such rings may be attached together in a fused    manner, and wherein each aryl may have 1 or more R^(x) substituents;-   wherein each heterocyclo is a saturated or partially saturated    heteroatom-containing ring radical, where the heteroatoms may be    selected from nitrogen, sulfur and oxygen, wherein each heterocyclo    is a monocyclic 6-8 membered ring or a 5-16 membered bicyclic ring    system, and wherein each heterocyclo may have 1 to 3 R^(x)    substituents;    or a pharmaceutically acceptable salt, prodrug or isotopic variant,    for example, partially or fully deuterated form thereof.

In some aspects, the compound is of Formula I or Formula II and R⁶ isabsent.

In some aspects, the compound is of Formula III:

and the variables are as defined for compounds of Formulae I and II andpharmaceutically acceptable salts thereof.

In some aspects, R^(x) is not further substituted.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valence, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring and whereinm is 0 or 1 and n is 0, 1 or 2.

In some aspects, R⁸ is hydrogen or C₁-C₃ alkyl.

In some aspects, R is hydrogen or C₁-C₃ alkyl.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴,-(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ any of which may beoptionally independently substituted with one or more R^(x) groups asallowed by valence, and wherein two R^(x) groups bound to the same oradjacent atom may optionally combine to form a ring.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴,-(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ without furthersubstitution.

In some aspects, m in R² is 1. In a further aspect, the alkylene in R²is methylene.

In some aspects, R² is

wherein:R^(2*) is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,-(alkylene)_(m)-C(O)-(alkylene)_(m)-,-(alkylene)_(m)-S(O)₂-(alkylene)_(m)-, or-(alkylene)_(m)-NH-(alkylene)_(m)- wherein each m is independently 0 or1;P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group;each R^(x1) is independently-(alkylene)_(m)-(C(O))_(m)-(alkylene)_(m)-(N(R^(N)))_(m)-(alkyl)_(m)wherein each m is independently 0 or 1 provided at least one m is 1,—(C(O))—O-alkyl, -(alkylene)_(m)-cycloalkyl wherein m is 0 or 1,—N(R^(N))-cycloalkyl, —C(O)-cycloalkyl, -(alkylene)_(m)-heterocyclylwherein m is 0 or 1, or —N(R^(N))-heterocyclyl, —C(O)-heterocyclyl,—S(O)₂-(alkylene)_(m) wherein m is 1 or 2, wherein:

-   -   R^(N) is H, C₁ to C₄ alkyl or C₁ to C₆ heteroalkyl, and    -   wherein two R^(x1) can, together with the atoms to which they        attach on P, which may be the same atom, form a ring; and        t is 0, 1 or 2.

In some aspects, each R^(x1) is only optionally substituted byunsubstituted alkyl, halogen or hydroxy.

In some aspects, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl.

In some aspects, at least one R^(x1) is -(alkylene)_(m)-heterocyclylwherein m is 0 or 1.

In some aspects, R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some aspects, R² is

In some aspects, R² is

In some aspects, R² is

wherein:R^(2*) is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,-(alkylene)_(m)-C(O)-(alkylene)_(m)-,-(alkylene)_(m)-S(O)₂-(alkylene)_(m)-, or-(alkylene)_(m)-NH-(alkylene)_(m)- wherein each m is independently 0 or1;P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group;P1 is a 4- to 6-membered monocyclic saturated heterocyclyl group;each R^(X2) is independently hydrogen or alkyl; ands is 0, 1 or 2.

In some aspects, R² is

In some aspects, P1 includes at least one nitrogen.

In some aspects, any alkylene in R²* in any previous aspect is notfurther substituted.

In some aspects, R² is selected from the structures depicted in FIGS.2-4.

In some aspects, R² is

In some aspects, the compound has general Formula I and morespecifically one of the general structures disclosed in FIGS. 5A-9Fwherein the variables are as previously defined.

In some aspects, the compound has general Formula Ia:

wherein R¹, R², R, R⁸, X and y are as previously defined.

In some embodiments, the compound has Formula Ia and R is alkyl.

In some embodiments, the compound has Formula Ia and R is H.

In some embodiments, the compound has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl andR^(2*) is as previously defined.

In some embodiments, the compound has Formula Ib:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ib and R is alkyl.

In some embodiments, the compound has Formula Ib and R is H.

In some embodiments, the compound has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the compound has Formula Ic:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ic and R is alkyl.

In some embodiments, the compound has Formula Ic and R is H.

In some embodiments, the compound has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the compound has Formula Id:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Id and R is alkyl.

In some embodiments, the compound has Formula Id and R is H.

In some embodiments, the compound has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the compound has Formula Ie:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ie and R is alkyl.

In some embodiments, the compound has Formula Ie and R is H.

In some embodiments, the compound has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the compound has Formula If:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula If and R is alkyl.

In some embodiments, the compound has Formula If and R is H.

In some embodiments, the compound has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the compound has Formula Ig:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ig and R is alkyl.

In some embodiments, the compound has Formula Ig and R is H.

In some embodiments, the compound has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the compound has Formula Ih:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ih and R is alkyl.

In some embodiments, the compound has Formula Ih and R is H.

In some embodiments, the compound has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the compound has Formula Ii:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ii and R is alkyl.

In some embodiments, the compound has Formula Ii and R is H.

In some embodiments, the compound has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R^(2*), R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R^(2*) is aspreviously defined.

In some embodiments, the compound has Formula Ij:

wherein R, R² and R⁸ are as previously defined.

In some embodiments, the compound has Formula Ij and R is alkyl.

In some embodiments, the compound has Formula Ij and R is H.

In some embodiments, the compound has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Ij and R is H, and X is CHand N.

In some embodiments, the compound has the structure:

In some embodiments, the compound has the structure Ik:

In some embodiments, the compound has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Il:

In some embodiments, the compound has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Im:

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula IIa:

In some embodiments, the compound has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula IIb:

In some embodiments, the compound has Formula IIb and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula IIb and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some aspects, the active compound is:

Further specific compounds that fall within the present invention andthat can be used in the disclosed methods of treatment and compositionsinclude, but are not limited to, the structures listed in Table 1 below.

TABLE 1 Structures of Tricyclic Lactams Structure Reference Structure A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

BB

CC

DD

EE

FF

GG

HH

II

JJ

KK

LL

MM

NN

OO

PP

QQ

RR

SS

TT

UU

VV

WW

XX

YY

ZZ

AAA

BBB

CCC

DDD

EEE

FFF

GGG

HHH

III

JJJ

KKK

LLL

MMM

NNN

OOO

PPP

QQQ

RRR

SSS

TTT

UUU

VVV

WWW

XXX

YYY

ZZZ

AAAA

BBBB

CCCC

DDDD

EEEE

FFFF

GGGG

HHHH

Isotopic Substitution

The present invention includes compounds and the use of compounds withdesired isotopic substitutions of atoms, at amounts above the naturalabundance of the isotope, i.e., enriched. Isotopes are atoms having thesame atomic number but different mass numbers, i.e., the same number ofprotons but a different number of neutrons. By way of general exampleand without limitation, isotopes of hydrogen, for example, deuterium(²H) and tritium (³H) may be used anywhere in described structures.Alternatively or in addition, isotopes of carbon, e.g., ¹³C and ¹⁴C, maybe used. A preferred isotopic substitution is deuterium for hydrogen atone or more locations on the molecule to improve the performance of thedrug. The deuterium can be bound in a location of bond breakage duringmetabolism (an α-deuterium kinetic isotope effect) or next to or nearthe site of bond breakage (a β-deuterium kinetic isotope effect).

Substitution with isotopes such as deuterium can afford certaintherapeutic advantages resulting from greater metabolic stability, suchas, for example, increased in vivo half-life or reduced dosagerequirements. Substitution of deuterium for hydrogen at a site ofmetabolic break down can reduce the rate of or eliminate the metabolismat that bond. At any position of the compound that a hydrogen atom maybe present, the hydrogen atom can be any isotope of hydrogen, includingprotium (¹H), deuterium (²H) and tritium (³H). Thus, reference herein toa compound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

The term “isotopically-labeled” analog refers to an analog that is a“deuterated analog”, a “¹³C-labeled analog,” or a“deuterated/¹³C-labeled analog.” The term “deuterated analog” means acompound described herein, whereby a H-isotope, i.e., hydrogen/protium(¹H), is substituted by a H-isotope, i.e., deuterium (²H). Deuteriumsubstitution can be partial or complete. Partial deuterium substitutionmeans that at least one hydrogen is substituted by at least onedeuterium. In certain embodiments, the isotope is 90, 95 or 99% or moreenriched in an isotope at any location of interest. In some embodimentsit is deuterium that is 90, 95 or 99% enriched at a desired location.

CDK-Replication Dependent Cells and Cyclin-Dependent Kinase Inhibitors

Tissue-specific stem cells and subsets of other resident proliferatingcells are capable of self-renewal, meaning that they are capable ofreplacing themselves throughout the adult mammalian lifespan throughregulated replication. Additionally, stem cells divide asymmetrically toproduce “progeny” or “progenitor” cells that in turn produce variouscomponents of a given organ. For example, in the hematopoietic system,the hematopoietic stem cells give rise to progenitor cells which in turngive rise to all the differentiated components of blood (e.g., whiteblood cells, red blood cells, and platelets) (See FIG. 1).

Certain proliferating cells, such as HSPCs, require the enzymaticactivity of the proliferative kinases cyclin-dependent kinase 4 (CDK4)and/or cyclin-dependent kinase 6 (CDK6) for cellular replication. Incontrast, the majority of proliferating cells in adult mammals (e.g.,the more differentiated blood-forming cells in the bone marrow) do notrequire the activity of CDK4 and/or CDK6 (i.e., CDK4/6). Thesedifferentiated cells can proliferate in the absence of CDK4/6 activityby using other proliferative kinases, such as cyclin-dependent kinase 2(CDK2) or cyclin-dependent kinase 1 (CDK1).

The current invention provides methods and tricyclic lactam compounds tominimize the effect of chemotherapeutic agent toxicity on CDK4/6replication dependent healthy cells, such as hematopoietic stem cellsand hematopoietic progenitor cells (together referred to as HSPCs),and/or renal epithelial cells, in subjects, typically humans, that willbe, are being, or have been exposed to the chemotherapeutic agent(typically a DNA-damaging agent).

The current invention further provides for tricyclic lactam compounds ofFormula I, II, III, IV, V, or VI, as described herein, apharmaceutically acceptable composition, salt, isotopic analog, orprodrug thereof. In addition, the current invention, provides for thecompounds listed in Table 1. The invention further includesadministering an effective amount of a selected compound of Formula I,II, III, IV, V, or VI, as described herein, a pharmaceuticallyacceptable composition, salt, isotopic analog, or prodrug thereof, whichprovides a G1-arrest of healthy cells, for example HSPCs and/or renalepithelial cells, in a subject prior to, during, or following thesubject's exposure to a chemotherapeutic agent, such as a DNA-damagingchemotherapeutic agent

In certain embodiments, the tricyclic lactam is a CDK4/6 inhibitor. Inone embodiment, the tricyclic lactam provides short term and transientprotection, allowing a significant portion of the cells to synchronouslyrenter the cell-cycle quickly following the cessation of thechemotherapeutic agent's effect, for example within less than about 24,30, 36, 40, or 48 hours. In one embodiment, the compound is selectedfrom the compounds described in Table 1 or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof. Cells that arequiescent within the G1 phase of the cell cycle are more resistant tothe damaging effect of chemotherapeutic agents than proliferating cells.

In one embodiment, a tricyclic lactam for use in the described methodsare CDK4/6 inhibitors, with minimal CDK2 inhibitory activity. In oneembodiment, a tricyclic lactam for use in the methods described hereinhas a CDK4/CycD1 IC₅₀ inhibitory concentration value thatis >100, >200, >300, >400, >500, >600, >700, >800, >900, >1000, >1250, >1500times, >1800 times, >2000 times, >2200 times, >2500 times, >2700times, >3000 times, >3200 times lower than its respective IC₅₀concentration value for CDK2/CycE inhibition. In one embodiment, atricyclic lactam for use in the methods described herein has an IC₅₀concentration value for CDK4/CycD1 inhibition that is about <1.50 nM,<1.25 nM, <1.0 nM, <0.90 nM, <0.85 nM, <0.80 nM, <0.75 nM, <0.70 nM,<0.65 nM, <0.60 nM, <0.55 nM, or less. In one embodiment, a tricycliclactam for use in the methods described herein has an IC₅₀ concentrationvalue for CDK2/CycE inhibition that is about >1.0 μM, >1.25 μM, >1.50μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50 μM, >2.75 μM, >3.0 μM, >3.25μM, >3.5 μM or greater. In one embodiment, a tricyclic lactam for use inthe methods described herein has an IC₅₀ concentration value forCDK2/CycA IC₅₀ that is >0.80 μM, >0.85 μM, >0.90 μM, >0.95 μM, >0.1.0μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50 μM, >2.75μM, >3.0 μM or greater. In one embodiment, the tricyclic lactam for usein the methods described herein are selected from the group consistingof Formula I, Formula II, Formula III, Formula IV, Formula V, andFormula VI, or a pharmaceutically acceptable composition, salt, orprodrug, thereof. In one embodiment, the compound is selected from thecompounds described in Table 1, or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof.

In one embodiment, the tricyclic lactams described herein are used inCDK4/6-replication dependent healthy cell cycling strategies wherein asubject is exposed to regular, repeated chemotherapeutic treatments,wherein the healthy cells are G1-arrested when the subject is exposed tothe chemotherapeutic agent and allowed to reenter the cell-cycle beforethe subject's next chemotherapeutic treatment. Such cycling allowsCDK4/6-replication dependent cells to regenerate damaged blood celllineages between regular, repeated treatments, for example thoseassociated with standard chemotherapeutic treatments for cancer, andreduces the risk associated with long term CDK4/6 inhibition.

Proliferative disorders that are treated with chemotherapy includecancerous and non-cancer diseases. In a typical embodiment, theproliferative disorder is a CDK4/6-replication independent disorder. Thetricylic lactam compounds may be effective in protecting healthyCDK4/6-replication dependent cells, for example HSPCs, duringchemotherapeutic treatment of a broad range of tumor types, includingbut not limited to the following: breast, prostate, ovarian, skin, lung,colorectal, brain (i.e., glioma) and renal. Preferably, the tricyliclactam should not compromise the efficacy of the chemotherapeutic agentor G1 arrest the cancer cells. Many cancers do not depend on theactivities of CDK4/6 for proliferation as they can use the proliferativekinases promiscuously (e.g., can use CDK 1/2/4/or 6) or lack thefunction of the retinoblastoma tumor suppressor protein (Rb), which isinactivated by the CDKs. The potential sensitivity of certain tumors toCDK4/6 inhibition can be deduced based on tumor type and moleculargenetics using standard techniques. Cancers that are not typicallyaffected by the inhibition of CDK4/6 are those that can be characterizedby one or more of the group including, but not limited to, increasedactivity of CDK1 or CDK2, loss, deficiency, or absence of retinoblastomatumor suppressor protein (Rb), high levels of MYC expression, increasedcyclin E (e.g., E1 or E2) and increased cyclin A, or expression of aRb-inactivating protein (such as HPV-encoded E7). Such cancers caninclude, but are not limited to, small cell lung cancer, retinoblastoma,HPV positive malignancies like cervical cancer and certain head and neckcancers, MYC amplified tumors such as Burkitts' Lymphoma, and triplenegative breast cancer; certain classes of sarcoma, certain classes ofnon-small cell lung carcinoma, certain classes of melanoma, certainclasses of pancreatic cancer, certain classes of leukemia, certainclasses of lymphoma, certain classes of brain cancer, certain classes ofcolon cancer, certain classes of prostate cancer, certain classes ofovarian cancer, certain classes of uterine cancer, certain classes ofthyroid and other endocrine tissue cancers, certain classes of salivarycancers, certain classes of thymic carcinomas, certain classes of kidneycancers, certain classes of bladder cancers, and certain classes oftesticular cancers.

The loss or absence of retinoblastoma (Rb) tumor suppressor protein(Rb-null) can be determined through any of the standard assays known toone of ordinary skill in the art, including but not limited to WesternBlot, ELISA (enzyme linked immunoadsorbent assay), IHC(immunohistochemistry), and FACS (fluorescent activated cell sorting).The selection of the assay will depend upon the tissue, cell line orsurrogate tissue sample that is utilized e.g., for example Western Blotand ELISA may be used with any or all types of tissues, cell lines orsurrogate tissues, whereas the IHC method would be more appropriatewherein the tissue utilized in the methods of the present invention wasa tumor biopsy. FACs analysis would be most applicable to samples thatwere single cell suspensions such as cell lines and isolated peripheralblood mononuclear cells. See for example, US 20070212736 “FunctionalImmunohistochemical Cell Cycle Analysis as a Prognostic Indicator forCancer”.

Alternatively, molecular genetic testing may be used for determinationof retinoblastoma gene status. Molecular genetic testing forretinoblastoma includes the following as described in Lohmann and Gallie“Retinoblastoma. Gene Reviews” (2010)http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=retinoblastomaor Parsam et al. “A comprehensive, sensitive and economical approach forthe detection of mutations in the RB 1 gene in retinoblastoma” Journalof Genetics, 88(4), 517-527 (2009).

Increased activity of CDK1 or CDK2, high levels of MYC expression,increased cyclin E and increased cyclin A can be determined through anyof the standard assays known to one of ordinary skill in the art,including but not limited to Western Blot, ELISA (enzyme linkedimmunoadsorbent assay), IHC (immunohistochemistry), and FACS(fluorescent activated cell sorting). The selection of the assay willdepend upon the tissue, cell line, or surrogate tissue sample that isutilized e.g., for example Western Blot and ELISA may be used with anyor all types of tissues, cell lines, or surrogate tissues, whereas theIHC method would be more appropriate wherein the tissue utilized in themethods of the present invention was a tumor biopsy. FACs analysis wouldbe most applicable to samples that were single cell suspensions such ascell lines and isolated peripheral blood mononuclear cells.

In some embodiments, the cancer is selected from a small cell lungcancer, retinoblastoma, and triple negative (ER/PR/Her2 negative) or“basal-like” breast cancer, which almost always inactivate theretinoblastoma tumor suppressor protein (Rb), and therefore do notrequire CDK4/6 activity to proliferate. Triple negative (basal-like)breast cancer is also almost always genetically or functionally Rb-null.Also, certain virally induced cancers (e.g. cervical cancer and subsetsof Head and Neck cancer) express a viral protein (E7) which inactivatesRb making these tumors functionally Rb-null. Some lung cancers are alsobelieved to be caused by HPV. In one particular embodiment, the canceris small cell lung cancer, and the patient is treated with aDNA-damaging agent selected from the group consisting of etoposide,carboplatin, and cisplatin, or a combination thereof.

The tricyclic lactams described herein can also be used in protectinghealthy CDK4/6-replication dependent cells during chemotherapeutictreatments of abnormal tissues in non-cancer proliferative diseases,including but not limited to: psoriasis, lupus, arthritis (notablyrheumatoid arthritis), hemangiomatosis in infants, multiple sclerosis,myelodegenerative disease, neurofibromatosis, ganglioneuromatosis,keloid formation, Paget's Disease of the bone, fibrocystic disease ofthe breast, Peyronie's and Duputren's fibrosis, restenosis, andcirrhosis. Further, a tricyclic lactam described herein can be used toameliorate the effects of chemotherapeutic agents in the event ofaccidental exposure or overdose (e.g., methotrexate overdose).

According to the present invention, the tricyclic lactam compound can beadministered to a subject on any chemotherapeutic treatment schedule andin any dose consistent with the prescribed course of treatment. Thetricyclic lactam compound is administered prior to, during, or followingthe administration of the chemotherapeutic agent. In one embodiment, thetricyclic lactams described herein can be administered to the subjectduring the time period ranging from 24 hours prior to chemotherapeutictreatment until 24 hours following exposure. This time period, however,can be extended to time earlier that 24 hour prior to exposure to theagent (e.g., based upon the time it takes the chemotherapeutic agentused to achieve suitable plasma concentrations and/or the compound'splasma half-life). Further, the time period can be extended longer than24 hours following exposure to the chemotherapeutic agent so long aslater administration of the tricyclic lactam leads to at least someprotective effect. Such post-exposure treatment can be especially usefulin cases of accidental exposure or overdose.

In some embodiments, the tricyclic lactam can be administered to thesubject at a time period prior to the administration of thechemotherapeutic agent, so that plasma levels of the tricyclic lactamare peaking at the time of administration of the chemotherapeutic agent.If convenient, the tricyclic lactam can be administered at the same timeas the chemotherapeutic agent, in order to simplify the treatmentregimen. In some embodiments, the chemoprotectant and chemotherapeuticcan be provided in a single formulation.

In some embodiments, the tricyclic lactam can be administered to thesubject such that the chemotherapeutic agent can be administered eitherat higher doses (increased chemotherapeutic dose intensity) or morefrequently (increased chemotherapeutic dose density). Dose-densechemotherapy is a chemotherapy treatment plan in which drugs are givenwith less time between treatments than in a standard chemotherapytreatment plan. Chemotherapy dose intensity represents unit dose ofchemotherapy administered per unit time. Dose intensity can be increasedor decreased through altering dose administered, time interval ofadministration, or both. Myelosuppression continues to represent themajor dose-limiting toxicity of cancer chemotherapy, resulting inconsiderable morbidity and mortality along with frequent reductions inchemotherapy dose intensity, which may compromise disease control andsurvival. The compounds and their use as described herein represent away of increasing chemotherapy dose density and/or dose intensity whilemitigating adverse events such as, but not limited to, myelosuppression.

If desired, multiple doses of the tricyclic lactam compound can beadministered to the subject. Alternatively, the subject can be given asingle dose of the tricyclic lactam.

In one embodiment, the tricyclic lactam can be administered so thatCDK4/6-replication dependent healthy cells are G1 arrested duringchemotherapeutic agent exposure and a significant number of healthycells reenter the cell-cycle and are capable of replicating shortlyafter chemotherapeutic agent exposure, for example, within about 24-48hours or less, and continue to replicate until administration of thetricyclic lactam in anticipation of the next chemotherapeutic treatment.In one embodiment, the tricyclic lactam is administered to allow for thecycling of the CDK4/6-replication dependent healthy cells betweenG1-arrest and reentry into the cell-cycle to accommodate arepeated-dosing chemotherapeutic treatment regimen, for example,including but not limited to a treatment regimen wherein thechemotherapeutic agent is administered: on day 1-3 every 21 days; ondays 1-3 every 28 days; on day 1 every 3 weeks; on day 1, day 8, and day15 every 28 days, on day 1 and day 8 every 28 days; on days 1 and 8every 21 days; on days 1-5 every 21 days; 1 day a week for 6-8 weeks; ondays 1, 22, and 43; days 1 and 2 weekly; days 1-4 and 22-25; 1-4; 22-25,and 43-46; and similar type-regimens, wherein the CDK4/6-replicationdependent cells are G1 arrested during chemotherapeutic agent exposureand a significant portion of the cells reenter the cell-cycle in betweenchemotherapeutic agent exposure.

In one embodiment, the tricyclic lactam described herein is used toprovide chemoprotection to a subject's CDK4/6-replication dependenthealthy cells during a CDK4/6-replication independent small cell lungcancer treatment protocol. In one embodiment, the tricyclic lactam isadministered to provide chemoprotection in a small cell lung cancertherapy protocol such as, but not limited to: cisplatin 60 mg/m2 IV onday 1 plus etoposide 120 mg/m2 IV on days 1-3 every 21 d for 4 cycles;cisplatin 80 mg/m2 IV on day 1 plus etoposide 100 mg/m2 IV on days 1-3every 28 d for 4 cycles; cisplatin 60-80 mg/m2 IV on day 1 plusetoposide 80-120 mg/m2 IV on days 1-3 every 21-28 d (maximum of 4cycles); carboplatin AUC 5-6 IV on day 1 plus etoposide 80-100 mg/m2 IVon days 1-3 every 28 d (maximum of 4 cycles); Cisplatin 60-80 mg/m2 IVon day 1 plus etoposide 80-120 mg/m2 IV on days 1-3 every 21-28 d;carboplatin AUC 5-6 IV on day 1 plus etoposide 80-100 mg/m2 IV on days1-3 every 28 d (maximum 6 cycles); cisplatin 60 mg/m2 IV on day 1 plusirinotecan 60 mg/m2 IV on days 1, 8, and 15 every 28 d (maximum 6cycles); cisplatin 30 mg/m2 IV on days 1 and 8 or 80 mg/m2 IV on day 1plus irinotecan 65 mg/m2 IV on days 1 and 8 every 21 d (maximum 6cycles); carboplatin AUC 5 IV on day 1 plus irinotecan 50 mg/m2 IV ondays 1, 8, and 15 every 28 d (maximum 6 cycles); carboplatin AUC 4-5 IVon day 1 plus irinotecan 150-200 mg/m2 IV on day 1 every 21 d (maximum 6cycles); cyclophosphamide 800-1000 mg/m2 IV on day 1 plus doxorubicin40-50 mg/m2 IV on day 1 plus vincristine 1-1.4 mg/m2 IV on day 1 every21-28 d (maximum 6 cycles); Etoposide 50 mg/m2 PO daily for 3 wk every 4wk; topotecan 2.3 mg/m2 PO on days 1-5 every 21 d; topotecan 1.5 mg/m2IV on days 1-5 every 21 d; carboplatin AUC 5 IV on day 1 plus irinotecan50 mg/m2 IV on days 1, 8, and 15 every 28 d; carboplatin AUC 4-5 IV onday 1 plus irinotecan 150-200 mg/m2 IV on day 1 every 21 d; cisplatin 30mg/m2 IV on days 1, 8, and 15 plus irinotecan 60 mg/m2 IV on days 1, 8,and 15 every 28 d; cisplatin 60 mg/m2 IV on day 1 plus irinotecan 60mg/m2 IV on days 1, 8, and 15 every 28 d; cisplatin 30 mg/m2 IV on days1 and 8 or 80 mg/m2 IV on day 1 plus irinotecan 65 mg/m2 IV on days 1and 8 every 21 d; paclitaxel 80 mg/m2 IV weekly for 6 wk every 8 wk;paclitaxel 175 mg/m2 IV on day 1 every 3 wk; etoposide 50 mg/m2 PO dailyfor 3 wk every 4 wk; topotecan 2.3 mg/m2 PO on days 1-5 every 21 d;topotecan 1.5 mg/m2 IV on days 1-5 every 21 d; carboplatin AUC 5 IV onday 1 plus irinotecan 50 mg/m2 IV on days 1, 8, and 15 every 28 d;carboplatin AUC 4-5 IV on day 1 plus irinotecan 150-200 mg/m2 IV on day1 every 21 d; cisplatin 30 mg/m2 IV on days 1, 8, and 15 plus irinotecan60 mg/m2 IV on days 1, 8, and 15 every 28 d; cisplatin 60 mg/m2 IV onday 1 plus irinotecan 60 mg/m2 IV on days 1, 8, and 15 every 28 d;cisplatin 30 mg/m2 IV on days 1 and 8 or 80 mg/m2 IV on day 1 plusirinotecan 65 mg/m2 IV on days 1 and 8 every 21 d; paclitaxel 80 mg/m2IV weekly for 6 wk every 8 wk; and paclitaxel 175 mg/m2 IV on day 1every 3 wk.

In one embodiment, a tricyclic lactam described herein is administeredto a subject with small cell lung cancer on days 1, 2, and 3 of atreatment protocol wherein the DNA damaging agent selected from thegroup consisting of carboplatin, etoposide, and cisplatin, or acombination thereof, is administered on days 1, 2, and 3 every 21 days.

In one embodiment, a tricyclic lactam described herein is used toprovide chemoprotection to a subject's CDK4/6-replication dependenthealthy cells during a CDK4/6-replication independent head and neckcancer treatment protocol. In one embodiment, the tricyclic lactam isadministered to provide chemoprotection in a CDK4/6-replicationindependent head and neck cancer therapy protocol such as, but notlimited to: cisplatin 100 mg/m2 IV on days 1, 22, and 43 or 40-50 mg/m2IV weekly for 6-7 wk; cetuximab 400 mg/m2 IV loading dose 1 wk beforethe start of radiation therapy, then 250 mg/m2 weekly (premedicate withdexamethasone, diphenhydramine, and ranitidine); cisplatin 20 mg/m2 IVon day 2 weekly for up to 7 wk plus paclitaxel 30 mg/m2 IV on day 1weekly for up to 7 wk; cisplatin 20 mg/m2/day IV on days 1-4 and 22-25plus 5-FU 1000 mg/m2/day by continuous IV infusion on days 1-4 and22-25; 5-FU 800 mg/m2 by continuous IV infusion on days 1-5 given on thedays of radiation plus hydroxyurea 1 g PO q12h (11 doses per cycle);chemotherapy and radiation given every other week for a total of 13 wk;carboplatin 70 mg/m2/day IV on days 1-4, 22-25, and 43-46 plus 5-FU 600mg/m2/day by continuous IV infusion on days 1-4, 22-25, and 43-46;carboplatin AUC 1.5 IV on day 1 weekly plus paclitaxel 45 mg/m2 IV onday 1 weekly; cisplatin 100 mg/m2 IV on days 1, 22, and 43 or 40-50mg/m2 IV weekly for 6-7 wk; docetaxel 75 mg/m2 IV on day 1 pluscisplatin 100 mg/m2 IV on day 1 plus 5-FU 100 mg/m2/day by continuous IVinfusion on days 1-4 every 3 wk for 3 cycles, then 3-8 wk later,carboplatin AUC 1.5 IV weekly for up to 7 wk during radiation therapy;docetaxel 75 mg/m2 IV on day 1 plus cisplatin 75 mg/m2 IV on day 1 plus5-FU 750 mg/m2/day by continuous IV infusion on days 1-4 every 3 wk for4 cycles; cisplatin 100 mg/m2 IV on day 1 every 3 wk for 6 cycles plus5-FU 1000 mg/m2/day by continuous IV infusion on days 1-4 every 3 wk for6 cycles plus cetuximab 400 mg/m2 IV loading dose on day 1, then 250mg/m2 IV weekly until disease progression (premedicate withdexamethasone, diphenhydramine, and ranitidine); carboplatin AUC 5 IV onday 1 every 3 wk for 6 cycles plus 5-FU 1000 mg/m2/day by continuous IVinfusion on days 1-4 every 3 wk for 6 cycles plus cetuximab 400 mg/m2 IVloading dose on day 1, then 250 mg/m2 IV weekly until diseaseprogression (premedicate with dexamethasone, diphenhydramine, andranitidine); cisplatin 75 mg/m2 IV on day 1 plus docetaxel 75 mg/m2 IVon day 1 every 3 wk; cisplatin 75 mg/m2 IV on day 1 plus paclitaxel 175mg/m2 IV on day 1 every 3 wk; carboplatin AUC 6 IV on day 1 plusdocetaxel 65 mg/m2 IV on day 1 every 3 wk; carboplatin AUC 6 IV on day 1plus paclitaxel 200 mg/m2 IV on day 1 every 3 wk; cisplatin 75-100 mg/m2IV on day 1 every 3-4 wk plus cetuximab 400 mg/m2 IV loading dose on day1, then 250 mg/m2 IV weekly (premedicate with dexamethasone,diphenhydramine, and ranitidine); cisplatin 100 mg/m2 IV on day 1 plus5-FU 1000 mg/m2/day by continuous IV infusion on days 1-4 every 3 wk;methotrexate 40 mg/m2 IV weekly (3 wk equals 1 cycle); paclitaxel 200mg/m2 IV every 3 wk; docetaxel 75 mg/m2 IV every 3 wk; cetuximab 400mg/m2 IV loading dose on day 1, then 250 mg/m2 IV weekly until diseaseprogression (premedicate with dexamethasone, diphenhydramine, andranitidine); cisplatin 100 mg/m2 IV on day 1 every 3 wk for 6 cyclesplus 5-FU 1000 mg/m2/day by continuous IV infusion on days 1-4 every 3wk for 6 cycles plus cetuximab 400 mg/m2 IV loading dose on day 1, then250 mg/m2 IV weekly (premedicate with dexamethasone, diphenhydramine,and ranitidine); carboplatin AUC 5 IV on day 1 every 3 wk for 6 cyclesplus 5-FU 1000 mg/m2/day by continuous IV infusion on days 1-4 every 3wk for 6 cycles plus cetuximab 400 mg/m2 IV loading dose on day 1, then250 mg/m2 IV weekly (premedicate with dexamethasone, diphenhydramine,and ranitidine); cisplatin 75 mg/m2 IV on day 1 plus docetaxel 75 mg/m2IV on day 1 every 3 wk; cisplatin 75 mg/m2 IV on day 1 plus paclitaxel175 mg/m2 IV on day 1 every 3 wk; carboplatin AUC 6 IV on day 1 plusdocetaxel 65 mg/m2 IV on day 1 every 3 wk; carboplatin AUC 6 IV on day 1plus paclitaxel 200 mg/m2 IV on day 1 every 3 wk; cisplatin 75-100 mg/m2IV on day 1 every 3-4 wk plus cetuximab 400 mg/m2 IV loading dose on day1, then 250 mg/m2 IV weekly (premedicate with dexamethasone,diphenhydramine, and ranitidine); cisplatin 100 mg/m2 IV on day 1 plus5-FU 1000 mg/m2/day by continuous IV infusion on days 1-4 every 3 wk;methotrexate 40 mg/m2 IV weekly (3 wk equals 1 cycle); paclitaxel 200mg/m2 IV every 3 wk; docetaxel 75 mg/m2 IV every 3 wk; cetuximab 400mg/m2 IV loading dose on day 1, then 250 mg/m2 IV weekly until diseaseprogression (premedicate with dexamethasone, diphenhydramine, andranitidine); cisplatin 100 mg/m2 IV on days 1, 22, and 43 withradiation, then cisplatin 80 mg/m2 IV on day 1 plus 5-FU 1000 mg/m2/dayby continuous IV infusion on days 1-4 every 4 wk for 3 cycles; cisplatin75 mg/m2 IV on day 1 plus docetaxel 75 mg/m2 IV on day 1 every 3 wk;cisplatin 75 mg/m2 IV on day 1 plus paclitaxel 175 mg/m2 IV on day 1every 3 wk; carboplatin AUC 6 IV on day 1 plus docetaxel 65 mg/m2 IV onday 1 every 3 wk; carboplatin AUC 6 IV on day 1 plus paclitaxel 200mg/m2 IV on day 1 every 3 wk; cisplatin 100 mg/m2 IV on day 1 plus 5-FU1000 mg/m2/day by continuous IV infusion on days 1-4 every 3 wk;cisplatin 50-70 mg/m2 IV on day 1 plus gemcitabine 1000 mg/m2 IV on days1, 8, and 15 every 4 wk; gemcitabine 1000 mg/m2 IV on days 1, 8, and 15every 4 wk or gemcitabine 1250 mg/m2 IV on days 1 and 8 every 3 wk;methotrexate 40 mg/m2 IV weekly (3 wk equals 1 cycle); paclitaxel 200mg/m2 IV every 3 wk; docetaxel 75 mg/m2 IV every 3 wk; cisplatin 75mg/m2 IV on day 1 plus docetaxel 75 mg/m2 IV on day 1 every 3 wk;cisplatin 75 mg/m2 IV on day 1 plus paclitaxel 175 mg/m2 IV on day 1every 3 wk; carboplatin AUC 6 IV on day 1 plus docetaxel 65 mg/m2 IV onday 1 every 3 wk; carboplatin AUC 6 IV on day 1 plus paclitaxel 200mg/m2 IV on day 1 every 3 wk; cisplatin 100 mg/m2 IV on day 1 plus 5-FU1000 mg/m2/day by continuous IV infusion on days 1-4 every 3 wk;cisplatin 50-70 mg/m2 IV on day 1 plus gemcitabine 1000 mg/m2 IV on days1, 8, and 15 every 4 wk; gemcitabine 1000 mg/m2 IV on days 1, 8, and 15every 4 wk or gemcitabine 1250 mg/m2 IV on days 1 and 8 every 3 wk;methotrexate 40 mg/m2 IV weekly (3 wk equals 1 cycle); paclitaxel 200mg/m2 IV every 3 wk; and docetaxel 75 mg/m2 IV every 3 wk.

In one embodiment, the tricyclic lactam described herein is used toprovide chemoprotection to a subject's CDK4/6-replication dependenthealthy cells during a CDK4/6-replication independent triple negativebreast cancer treatment protocol. In one embodiment, the tricycliclactam is administered to provide chemoprotection in aCDK4/6-replication independent triple negative breast cancer therapyprotocol such as, but not limited to: dose-dense doxorubicin(adriamycin) and cyclophosphamide (cytoxan) every two weeks for fourcycles followed by dose-dense paclitaxel (Taxol) every two weeks forfour cycles; adriamycin/paclitaxel/cyclophosphomide every three weeksfor a total of four cycles; adriamycin/paclitaxel/cyclophosphomide everytwo weeks for a total of four cycles; adriamycin/cyclophosphomidefollowed by paclitaxel (Taxol) every three weeks for four cycles each;and adriamycin/cyclophosphomide followed by paclitaxel (Taxol) every twoweeks for four cycles each.

Triple-negative breast cancer (TNBC) is defined as the absence ofstaining for estrogen receptor, progesterone receptor, and HER2/neu.TNBC is insensitive to some of the most effective therapies availablefor breast cancer treatment including HER2-directed therapy such astrastuzumab and endocrine therapies such as tamoxifen or the aromataseinhibitors. Combination cytotoxic chemotherapy administered in adose-dense or metronomic schedule remains the standard therapy forearly-stage TNBC. Platinum agents have recently emerged as drugs ofinterest for the treatment of TNBC with carboplatin added to paclitaxeland adriamycin plus cyclophosphamide chemotherapy in the neoadjuvantsetting. The poly (ADP-ribose) polymerase (PARP) inhibitors are emergingas promising therapeutics for the treatment of TNBC. PARPs are a familyof enzymes involved in multiple cellular processes, including DNArepair.

As a nonlimiting illustration, the subject is exposed tochemotherapeutic agent at least 5 times a week, at least 4 times a week,at least 3 times a week, at least 2 times a week, at least 1 time aweek, at least 3 times a month, at least 2 times a month, or at least 1time a month, wherein the subject's CDK4/6-replication dependent healthycells are G1 arrested during treatment and allowed to cycle in betweenchemotherapeutic agent exposure, for example during a treatment break.In one embodiment, the subject is undergoing 5 times a weekchemotherapeutic treatment, wherein the subject's CDK4/6-replicationdependent healthy cells are G1 arrested during the chemotherapeuticagent exposure and allowed to reenter the cell-cycle during the 2 daybreak, for example, over the weekend.

In one embodiment, using a tricyclic lactam described herein, thesubject's CDK4/6-replicaton dependent healthy cells are arrested duringthe entirety of the chemotherapeutic agent exposure time-period, forexample, during a contiguous multi-day regimens, the cells are arrestedover the time period that is required to complete the contiguousmulti-day course, and then allowed to recycle at the end of thecontiguous multi-day course. In one embodiment, using a tricyclic lactamdescribed herein, the subject's CDK4/6-replication dependent healthycells are arrested during the entirety of the chemotherapeutic regimen,for example, in a daily chemotherapeutic exposure for three weeks, andrapidly reenter the cell-cycle following the completion of thetherapeutic regimen.

In one embodiment, the subject has been exposed to a chemotherapeuticagent, and, using a tricyclic lactam described herein, the subject'sCDK4/6-replication dependent healthy cells are placed in G1 arrestfollowing exposure in order to mitigate, for example, DNA damage. In oneembodiment, the tricyclic lactam is administered at least ½ hour, atleast 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, atleast 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, atleast 10 hours, at least 12 hours, at least 14 hours, at least 16 hours,at least 18 hours, at least 20 hours or more post chemotherapeutic agentexposure.

In one embodiment, a tricyclic lactam can allow for dose intensification(e.g., more therapy can be given in a fixed period of time) in medicallyrelated chemotherapies, which will translate to better efficacy.Therefore, the presently disclosed methods can result in chemotherapyregimens that are less toxic and more effective.

The use of a tricyclic lactam as described herein can induce selectiveG1 arrest in CDK4/6-dependent cells (e.g., as measured in a cell-basedin vitro assay). In one embodiment, the tricyclic lactam is capable ofincreasing the percentage of CDK4/6-dependent cells in the G1 phase,while decreasing the percentage of CDK4/6-dependent cells in the G2/Mphase and S phase. In one embodiment, the tricyclic lactam inducessubstantially pure (i.e., “clean”) G1 cell cycle arrest in theCDK4/6-dependent cells (e.g., wherein treatment with the tricycliclactam induces cell cycle arrest such that the majority of cells arearrested in G1 as defined by standard methods (e.g. propidium iodide(PI) staining or others) with the population of cells in the G2/M and Sphases combined being less than about 30%, about 25%, about 20%, about15%, about 10%, about 5%, about 3% or less of the total cell population.Methods of assessing the cell phase of a population of cells are knownin the art (see, for example, in U.S. Patent Application Publication No.2002/0224522) and include cytometric analysis, microscopic analysis,gradient centrifugation, elutriation, fluorescence techniques includingimmunofluorescence, and combinations thereof. Cytometric techniquesinclude exposing the cell to a labeling agent or stain, such asDNA-binding dyes, e.g., PI, and analyzing cellular DNA content by flowcytometry. Immunofluorescence techniques include detection of specificcell cycle indicators such as, for example, thymidine analogs (e.g.,5-bromo-2-deoxyuridine (BrdU) or an iododeoxyuridine), with fluorescentantibodies.

In some embodiments, the use of a tricyclic lactam described herein mayresult in reduced or substantially free of off-target effects, forexample, related to inhibition of kinases other than CDK4 and/or CDK6such as CDK2. Furthermore, in certain embodiments, the use of thecompounds described herein should not induce cell cycle arrest in CDK4/6replication independent cells.

In some embodiments, the use of a tricyclic lactam described hereinreduces the risk of undesirable off-target effects including, but notlimited to, long term toxicity, anti-oxidant effects, and estrogeniceffects. Anti-oxidant effects can be determined by standard assays knownin the art. For example, a compound with no significant anti-oxidanteffects is a compound that does not significantly scavengefree-radicals, such as oxygen radicals. The anti-oxidant effects of acompound can be compared to a compound with known anti-oxidant activity,such as genistein. Thus, a compound with no significant anti-oxidantactivity can be one that has less than about 2, 3, 5, 10, 30, or 100fold anti-oxidant activity relative to genistein. Estrogenic activitiescan also be determined via known assays. For instance, a non-estrogeniccompound is one that does not significantly bind and activate theestrogen receptor. A compound that is substantially free of estrogeniceffects can be one that has less than about 2, 3, 5, 10, 20, or 100 foldestrogenic activity relative to a compound with estrogenic activity,e.g., genistein.

Active Compounds, Salts, and Formulations

As used herein, the term “active compound” refers to the tricycliclactam compounds described herein or a pharmaceutically acceptable salt,isotopic analog, or prodrug thereof. The active compound can beadministered to the subject through any suitable approach. The amountand timing of active compound administered can, of course, be dependenton the subject being treated, on the dosage of chemotherapy to which thesubject is anticipated of being exposed to, on the time course of thechemotherapeutic agent exposure, on the manner of administration, on thepharmacokinetic properties of the particular active compound, and on thejudgment of the prescribing physician. Thus, because of subject tosubject variability, the dosages given below are a guideline and thephysician can titrate doses of the compound to achieve the treatmentthat the physician considers appropriate for the subject. In consideringthe degree of treatment desired, the physician can balance a variety offactors such as age and weight of the subject, presence of preexistingdisease, as well as presence of other diseases. Pharmaceuticalformulations can be prepared for any desired route of administrationincluding, but not limited to, oral, intravenous, or aerosoladministration, as discussed in greater detail below.

The therapeutically effective dosage of any active compound describedherein will be determined by the health care practitioner depending onthe condition, size and age of the patient as well as the route ofdelivery. In one non-limited embodiment, a dosage from about 0.1 toabout 200 mg/kg has therapeutic efficacy, with all weights beingcalculated based upon the weight of the active compound, including thecases where a salt is employed. In some embodiments, the dosage can bethe amount of compound needed to provide a serum concentration of theactive compound of up to between about 1 and 5, 10, 20, 30, or 40 μM. Insome embodiments, a dosage from about 10 mg/kg to about 50 mg/kg can beemployed for oral administration. Typically, a dosage from about 0.5mg/kg to 5 mg/kg can be employed for intramuscular injection. In someembodiments, dosages can be from about 1 μmol/kg to about 50 μmol/kg,or, optionally, between about 22 μmol/kg and about 33 μmol/kg of thecompound for intravenous or oral administration. An oral dosage form caninclude any appropriate amount of active material, including for examplefrom 5 mg to, 50, 100, 200, or 500 mg per tablet or other solid dosageform.

In accordance with the presently disclosed methods, pharmaceuticallyactive compounds as described herein can be administered orally as asolid or as a liquid, or can be administered intramuscularly,intravenously, or by inhalation as a solution, suspension, or emulsion.In some embodiments, the compounds or salts also can be administered byinhalation, intravenously, or intramuscularly as a liposomal suspension.When administered through inhalation the active compound or salt can bein the form of a plurality of solid particles or droplets having anydesired particle size, and for example, from about 0.01, 0.1 or 0.5 toabout 5, 10, 20 or more microns, and optionally from about 1 to about 2microns. Compounds as disclosed in the present invention havedemonstrated good pharmacokinetic and pharmacodynamics properties, forinstance when administered by the oral or intravenous routes.

In one embodiment of the invention, these tricyclic lactam can beadministered in a concerted regimen with a blood growth factor agent. Assuch, in one embodiment, the use of the compounds and methods describedherein is combined with the use of hematopoietic growth factorsincluding, but not limited to, granulocyte colony stimulating factor(G-CSF, for example, sold as Neupogen (filgrastin), Neulasta(peg-filgrastin), or lenograstin), granulocyte-macrophage colonystimulating factor (GM-CSF, for example sold as molgramostim andsargramostim (Leukine)), M-CSF (macrophage colony stimulating factor),thrombopoietin (megakaryocyte growth development factor (MGDF), forexample sold as Romiplostim and Eltrombopag) interleukin (IL)-12,interleukin-3, interleukin-11 (adipogenesis inhibiting factor oroprelvekin), SCF (stem cell factor, steel factor, kit-ligand, or KL) anderythropoietin (EPO), and their derivatives (sold as for exampleepoetin-α as Darbopoetin, Epocept, Nanokine, Epofit, Epogin, Eprex andProcrit; epoetin-β sold as for example NeoRecormon, Recormon andMicera), epoetin-delta (sold as for example Dynepo), epoetin-omega (soldas for example Epomax), epoetin zeta (sold as for example Silapo andReacrit) as well as for example Epocept, EPOTrust, Erypro Safe,Repoeitin, Vintor, Epofit, Erykine, Wepox, Espogen, Relipoeitin,Shanpoietin, Zyrop and EPIAO).

The pharmaceutical formulations can comprise an active compounddescribed herein or a pharmaceutically acceptable salt thereof, in anypharmaceutically acceptable carrier. If a solution is desired, water maybe the carrier of choice for water-soluble compounds or salts. Withrespect to the water-soluble compounds or salts, an organic vehicle,such as glycerol, propylene glycol, polyethylene glycol, or mixturesthereof, can be suitable. In the latter instance, the organic vehiclecan contain a substantial amount of water. The solution in eitherinstance can then be sterilized in a suitable manner known to those inthe art, and for illustration by filtration through a 0.22-micronfilter. Subsequent to sterilization, the solution can be dispensed intoappropriate receptacles, such as depyrogenated glass vials. Thedispensing is optionally done by an aseptic method. Sterilized closurescan then be placed on the vials and, if desired, the vial contents canbe lyophilized.

In addition to the active compounds or their salts, the pharmaceuticalformulations can contain other additives, such as pH-adjustingadditives. In particular, useful pH-adjusting agents include acids, suchas hydrochloric acid, bases or buffers, such as sodium lactate, sodiumacetate, sodium phosphate, sodium citrate, sodium borate, or sodiumgluconate. Further, the formulations can contain antimicrobialpreservatives. Useful antimicrobial preservatives include methylparaben,propylparaben, and benzyl alcohol. An antimicrobial preservative istypically employed when the formulation is placed in a vial designed formulti-dose use. The pharmaceutical formulations described herein can belyophilized using techniques well known in the art.

For oral administration a pharmaceutical composition can take the formof solutions, suspensions, tablets, pills, capsules, powders, and thelike. Tablets containing various excipients such as sodium citrate,calcium carbonate and calcium phosphate may be employed along withvarious disintegrants such as starch (e.g., potato or tapioca starch)and certain complex silicates, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate,and talc are often very useful for tableting purposes. Solidcompositions of a similar type may be employed as fillers in soft andhard-filled gelatin capsules. Materials in this connection also includelactose or milk sugar as well as high molecular weight polyethyleneglycols. When aqueous suspensions and/or elixirs are desired for oraladministration, the compounds of the presently disclosed subject mattercan be combined with various sweetening agents, flavoring agents,coloring agents, emulsifying agents and/or suspending agents, as well assuch diluents as water, ethanol, propylene glycol, glycerin and variouslike combinations thereof.

In yet another embodiment of the subject matter described herein, thereis provided an injectable, stable, sterile formulation comprising anactive compound as described herein, or a salt thereof, in a unit dosageform in a sealed container. The compound or salt is provided in the formof a lyophilizate, which is capable of being reconstituted with asuitable pharmaceutically acceptable carrier to form a liquidformulation suitable for injection thereof into a subject. When thecompound or salt is substantially water-insoluble, a sufficient amountof emulsifying agent, which is physiologically acceptable, can beemployed in sufficient quantity to emulsify the compound or salt in anaqueous carrier. Particularly useful emulsifying agents includephosphatidyl cholines and lecithin.

Additional embodiments provided herein include liposomal formulations ofthe active compounds disclosed herein. The technology for formingliposomal suspensions is well known in the art. When the compound is anaqueous-soluble salt, using conventional liposome technology, the samecan be incorporated into lipid vesicles. In such an instance, due to thewater solubility of the active compound, the active compound can besubstantially entrained within the hydrophilic center or core of theliposomes. The lipid layer employed can be of any conventionalcomposition and can either contain cholesterol or can becholesterol-free. When the active compound of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt can be substantially entrained within thehydrophobic lipid bilayer that forms the structure of the liposome. Ineither instance, the liposomes that are produced can be reduced in size,as through the use of standard sonication and homogenization techniques.The liposomal formulations comprising the active compounds disclosedherein can be lyophilized to produce a lyophilizate, which can bereconstituted with a pharmaceutically acceptable carrier, such as water,to regenerate a liposomal suspension.

Pharmaceutical formulations also are provided which are suitable foradministration as an aerosol by inhalation. These formulations comprisea solution or suspension of a desired compound described herein or asalt thereof, or a plurality of solid particles of the compound or salt.The desired formulation can be placed in a small chamber and nebulized.Nebulization can be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the compounds or salts. The liquid droplets or solidparticles may for example have a particle size in the range of about 0.5to about 10 microns, and optionally from about 0.5 to about 5 microns.The solid particles can be obtained by processing the solid compound ora salt thereof, in any appropriate manner known in the art, such as bymicronization. Optionally, the size of the solid particles or dropletscan be from about 1 to about 2 microns. In this respect, commercialnebulizers are available to achieve this purpose. The compounds can beadministered via an aerosol suspension of respirable particles in amanner set forth in U.S. Pat. No. 5,628,984, the disclosure of which isincorporated herein by reference in its entirety.

When the pharmaceutical formulation suitable for administration as anaerosol is in the form of a liquid, the formulation can comprise awater-soluble active compound in a carrier that comprises water. Asurfactant can be present, which lowers the surface tension of theformulation sufficiently to result in the formation of droplets withinthe desired size range when subjected to nebulization.

The term “pharmaceutically acceptable salts” as used herein refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with subjects (e.g., human subjects) withoutundue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the presently disclosed subject matter.

Thus, the term “salts” refers to the relatively non-toxic, inorganic andorganic acid addition salts of compounds of the presently disclosedsubject matter. These salts can be prepared in situ during the finalisolation and purification of the compounds or by separately reactingthe purified compound in its free base form with a suitable organic orinorganic acid and isolating the salt thus formed. Pharmaceuticallyacceptable base addition salts may be formed with metals or amines, suchas alkali and alkaline earth metal hydroxides, or of organic amines.Examples of metals used as cations, include, but are not limited to,sodium, potassium, magnesium, calcium, and the like. Examples ofsuitable amines include, but are not limited to,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine, and procaine.

Salts can be prepared from inorganic acids sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide such as hydrochloric, nitric,phosphoric, sulfuric, hydrobromic, hydriodic, phosphorus, and the like.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, lactobionate, laurylsulphonate and isethionate salts,and the like. Salts can also be prepared from organic acids, such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids,aliphatic and aromatic sulfonic acids, etc. and the like. Representativesalts include acetate, propionate, caprylate, isobutyrate, oxalate,malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Pharmaceuticallyacceptable salts can include cations based on the alkali and alkalineearth metals, such as sodium, lithium, potassium, calcium, magnesium andthe like, as well as non-toxic ammonium, quaternary ammonium, and aminecations including, but not limited to, ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. Also contemplated are the saltsof amino acids such as arginate, gluconate, galacturonate, and the like.See, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19, which isincorporated herein by reference.

Synthesis of Tricyclic Lactams

Tricyclic lactams of the present invention can be synthesized by anymeans known to those of ordinary skill in the art, including forexample, according to the generalized Schemes of 1 through 11 below.

A method for the preparation of substituted tricyclic lactams isprovided that includes efficient methods for the preparation of atricyclic lactam ring system and subsequent displacement of an arylsulfone with an amine.

In Scheme 1, diethyl succinate is employed to prepare the pyrimidineester, 2, according to the method of A. Haidle, See, WO 2009/152027entitled 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives forMARK inhibition. The ester intermediate 2 can be reduced by directlyreacting the ester with a reducing agent such as lithium borohydride ina protic organic solvent such as ethanol to produce the correspondingprimary alcohol. The primary alcohol can be reacted with a reagent suchas phosphorus tribromide in an organic solvent such as dimethylforamideto produce the primary bromide 3. The primary bromide 3 can be condensedwith the lactam 4 optionally at low temperature using a base such aslithium diisopropylamide in an organic solvent such as tetrahydrofuranto produce the lactam 5. Lactam 5 can be deprotected by directlyreacting Compound 5 with an aqueous acid such as HCl=pH 1 solution.Lactam 6 can be directly reacted with an organic base such as1,8-diazabicyclo[5.4.0]undec-7-ene in a protic solvent such as ethanoloptionally at high temperature to cyclize Compound 5 to form thetricyclic lactam 7. The thiol moiety can be subsequently oxidized to thesulfone 8 by directly reacting Compound 7 with an oxidizing reagent suchas meta-chloroperoxybenzoic acid. The sulfone, 8, can be directlyreacted with an amine, 9, in the presence of a strong base such aslithium hexamethyldisilazane to form the tricyclic lactam 10.

In Scheme 2, the tricyclic lactam 7 is directly reacted with anoxidizing reagent such as 2,3-dichloro-5,6-dicyano-1,4-benzoquinone(DDQ) to form the alkene 11. Alkene 11 can be directly reacted with anoxidizing reagent such as meta-chloroperoxybenzoic acid to form thesulfone intermediate 12. The sulfone, 12, can be condensed with anamine, 13, in the presence of a strong base such as lithiumhexamethyldisilazane to form the tricyclic lactam 14.

Scheme 3 illustrates the synthesis of a di-protected lactam useful inthe preparation of tricyclic lactams. Compound 15 is prepared accordingto the method of Arigon, J., See, US 2013/0289031 entitled Pyrimidinonederivatives, preparation thereof and pharmaceutical use thereof.Compound 15 is protected with a suitable protecting group by directlyreacting Compound 15 with di-tert-butyl carbonate (Boc anhydride) in thepresence of an organic base such as triethylamine ordiisopropylethylamine in an organic solvent such as dichloromethane toform the protected amine 16. The protected amine 16 can be directlyreacted with methyl chloroacetate in the presence of a base such aspotassium carbonate in an organic solvent such as acetonitrile to formthe ester 17. The ester 17 can be cyclized by directly reacting theester with an acid such as hydrochloric acid in a protic solvent such asmethanol optionally at a high temperature to form the spirolactam 18.The lactam 18 can be directly reacted with a protecting reagent such aschloromethyl methyl ether (MOM-Cl) in the presence of an organic basesuch as diisopropylethylamine in an organic solvent such asdichloromethane optionally at a low or at ambient temperature to formthe MOM-protected amine 19. The lactam 19 can be protected by directlyreacting the lactam with a suitable protecting reagent such aschloromethyl methyl ether (MOM-Cl) in the presence of a base such assodium bis(trimethylsilyl)amide in an organic solvent such astetrahydrofuran optionally at a low temperature. Additional lactamintermediates such as Compounds 25 and 31 can be synthesized usinganalogous chemistry as described for the synthesis of Compound 4. Thechemistry for the production of Compounds 25 and 31 is illustrated inSchemes 5 and 6.

Scheme 4 illustrates the coupling of a tricyclic lactam sulfone with anamine to generate compounds of Formula I, II, III, and IV.

Scheme 7 illustrates the preparation of the tricyclic lactam compound33. Compound 32 is prepared according to the method of Tavares, See,U.S. Pat. No. 8,598,186. Compound 32 is directly reacted with sulfone 8optionally in the presence of an organic base such as lithiumhexamethyldisilazane to form the amine 33. The same chemistry can beemployed to produce the alkene compound 34.

Other amine intermediates and final amine compounds can be synthesizedby those skilled in the art. It will be appreciated that the chemistrycan employ reagents that comprise reactive functionalities that can beprotected and de-protected and will be known to those skilled in the artat the time of the invention. See for example, Greene, T. W. and Wuts,P. G. M., Greene's Protective Groups in Organic Synthesis, 4^(th)edition, John Wiley and Sons.

In one embodiment a lactam intermediate is treated with BOC-anhydride inthe presence of an organic base such as triethylamine in an organicsolvent such as dichloromethane. The Boc protected lactam is treatedwith carbon dioxide in the presence of a nickel catalyst to generate acarboxylic acid. The carboxylic acid is reacted with thionyl chloride inthe presence of an organic solvent such as toluene. The resulting acidchloride is treated with an amine to generate an amide that can bedeprotected with a strong acid such as trifluoroacetic acid to generatethe final target compound.

Alternatively, the lactam can be generated by reacting the carboxylicacid with a protected amine in the presence of a strong acid and adehydrating agent, which can be together in one moiety as a strong acidanhydride. Examples of strong acid anhydrides include, but are notlimited to, trifluoroacetic acid anhydride, tribromoacetic acidanhydride, trichloroacetic acid anhydride, or mixed anhydrides. Thedehydrating agent can be a carbodiimide based compound such as but notlimited to DCC (N,N-dicyclohexylcarbodiimide), EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or DIC(N,N-diisopropylcarbodiimide). An additional step may be necessary totake off the N-protecting group and the methodologies are known to thoseskilled in the art

Alternatively, the SMe moiety bonded to the pyrimidine ring can besubstituted with any leaving group that can be displaced by a primaryamine, for example to create an intermediate for a final product such asBr, I, F, SO₂Me, SOalkyl, SO₂alkyl. See, for Example, PCT/US2013/037878to Tavares.

Other amine intermediates and final amine compounds can be synthesizedby those skilled in the art. It will be appreciated that the chemistrycan employ reagents that comprise reactive functionalities that can beprotected and de-protected and will be known to those skilled in the artat the time of the invention. See for example, Greene, T. W. and Wuts,P. G. M., Greene's Protective Groups in Organic Synthesis, 4^(th)edition, John Wiley and Sons.

[4-Chloro-2-(methylthio)pyrimidin-5-yl]methanol

4-Chloro-2-methylsulfanyl-5-pyrimidinecarboxylate ethyl ester (62 g, 260mmol) was dissolved in anhydrous tetrahydrofuran (500 mL) in a 3-necked5 L round bottomed flask fitted with a mechanical stirrer, additionfunnel, temperature probe and nitrogen inlet. The solution was cooled to0° C. Diisobutylaluminum hydride in tetrahydrofuran (1M solution, 800mL) was added dropwise over a period of 2 hours. After the addition wascomplete, the reaction mixture was kept at 0° C. for 0.5 hours. Thereaction was quenched at 0° C. by the slow addition of saturated aqueoussodium sulfate (265.3 mL, 530.7 mmol) keeping the internal reactiontemperature below 10° C. Ethyl acetate (900 mL) was added and thereaction slowly warmed to room temperature overnight. 6M HCl was addedtill the reaction mixture was slightly acidic (pH 6). The reactionmixture was filtered thru a pad of Celite® and the aluminum salts werewashed with ethyl acetate (1 L). The filtrate was poured into aseparatory funnel and washed twice with water (600 mL) and finally withbrine (600 mL). The organic layer was dried over sodium sulfate,filtered thru Celite® and the solvent concentrated in vacuo to afford39.2 g (77% crude yield) of a dark yellow oil. The material was used asis for the next step. NMR (CDCl₃) δ 8.56 (s, 1H), 4.76 (s, 2H), 2.59 (s,3H); MS (ESI+) for C₆H₇ClN₂OS m/z 191.0 (M+H)⁺.

4-Chloro-2-(methylthio)pyrimidine-5-carbaldehyde

[4-Chloro-2-(methylthio)pyrimidin-5-yl]methanol (39.2 g, 206 mmol) wastaken up in methylene chloride (520 mL) at room temperature.Manganese(IV) oxide (140 g, 1.60 mol) was added in one portion and thereaction mixture stirred at room temperature overnight. The reactionmixture was filtered through a pad of Celite® and washed with methylenechloride. The filtrate was concentrated under reduced pressure to afforda dark yellow semisolid. The crude product was purified by reverse phasechromatography running a gradient of 1:9 acetonitrile:water (0.1% TFA)to 100% acetonitrile (0.1% TFA). The desired fractions were combined andthe acetonitrile was removed under reduced pressure causingprecipitation of the desired product. The solids were removed byfiltration and the solids washed with water and dried under vacuum at50° C. Affords 16.6 g (43% yield) of the desired product as a whitesolid. NMR (CDCl₃) δ 10.32 (s, 1H), 8.88 (s, 1H), 2.65 (s, 3H); MS(ESI+) for C₆H₅ClN₂OS m/z 189.0 (M+H)⁺.

General Procedure A

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate

Isopropylmagnesium chloride:lithium chloride complex (4.43 g, 30.5 mmol,25.4 mL of a 1.2M solution) was added to tetrahydrofuran (104 mL) in a500 mL round bottomed flask which had been flame dried and cooled underArgon. The solution was cooled to −15° C. Ethyl propiolate (3.26 mL,32.1 mmol) was added dropwise affording a yellow solution. Stirring wascontinued at −15° C. for 30 minutes and then4-Chloro-2-(methylthio)pyrimidine-5-carbaldehyde (6.06 g, 32.1 mmol) intetrahydrofuran (52 mL) was added rapidly. After 10 minutes, thereaction was quenched by the addition of saturated aqueous ammoniumchloride (40 mL). The reaction mixture was warmed to room temperatureand poured into a separatory funnel partitioning between ethyl acetate(200 mL) and water (100 mL). The organic layer removed and the aqueouslayer extracted with ethyl acetate (100 mL). The combined organic layerswere washed with brine (100 mL), dried over sodium sulfate, filtered andthe solvent removed in vacuo to afford a dark red oil. The product waspurified by silica gel chromatography using a gradient of 1:4 to 2:3ethyl acetate:hexanes which afforded 3.76 g (37% yield) of the desiredproduct as a light red oil. NMR (CDCl₃) δ 8.75 (s, 1H), 5.81 (d, 1H,J=6.0 Hz), 2.72 (bs, 1H), 2.60 (s, 3H), 1.33 (t, 3H, J=7.2 Hz); MS(ESI+) for C₁₁H₁₁ClN₂O₃S m/z 287.9 (M+H)⁺.

tert-Butyl4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate

Following General Procedure A and using tert-butyl propiolate affordsthe desired product in 74% yield as a viscous yellow oil. NMR (CDCl₃) δ8.75 (s, 1H), 5.79 (d, 1H, J=5.4 Hz), 4.27 (q, 2H, J=7.2 Hz), 2.97 (d,1H, J=5.4 Hz), 2.60 (s, 3H), 1.52 (s, 9H); MS (ESI+) for C₁₃H₁₅ClN₂O₃Sm/z 314.9 (M+H)⁺.

General Procedure B

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate(3.40 g, 11.8 mmol) was taken up in 1,4-Dioxane (100 mL) at roomtemperature under argon. Triethylamine (3.3 mL, 24 mmol) was added andthe mixture heated to 60° C. for 1 h. The reaction mixture was cooled toroom temperature and the solvent removed in vacuo. The resultant darkorange oil was re-evaporated twice with toluene. Affords the desiredproduct (3:1 E:Z double bond isomers) in 99% yield as a dark orange oil.NMR (CDCl₃) (major E isomer) δ 8.69 (s, 1H), 7.65 (d, 1H, J=18.0 Hz),6.81 (d, 1H, J=18.0 Hz), 4.32 (q, 2H, J=6.0 Hz), 2.64 (s, 3H), 1.36 (t,3H, J=6.0 Hz); NMR (CDCl₃) (minor Z isomer) δ 8.87 (s, 1H), 6.89 (d, 1H,J=12.0 Hz), 6.23 (d, 1H, J=12.0 Hz), 4.14 (q, 2H, J=6.0 Hz), 2.63 (s,3H), 1.23 (t, 3H, J=6.0 Hz); MS (ESI+) for C₁₁H₁₁ClN₂O₃S m/z 287.0(M+H)⁺.

tert-Butyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate

Isomerization of tert-butyl4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-hydroxybut-2-ynoate usingGeneral Procedure B affords the desired product (5:1 E:Z double bondisomers) in a 99% yield as a viscous dark yellow oil. NMR (CDCl₃) (majorE isomer) δ 8.67 (s, 1H), 7.54 (d, 1H, J=15.6 Hz), 6.72 (d, 1H, J=15.6Hz), 2.64 (s, 3H), 1.54 (s, 9H); NMR (CDCl₃) (minor Z isomer) δ 8.86 (s,1H), 6.76 (d, 1H, J=12.0 Hz), 6.18 (d, 1H, J=12.0 Hz), 2.63 (s, 3H),1.40 (s, 9H); MS (ESI+) for C₁₃H₁₅ClN₂O₃S m/z 314.9 (M+H)⁺.

General Procedure C

Ethyl2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Ethyl 4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate (3.40g, 11.8 mmol) was taken up in acetonitrile (20 mL) at room temperature.1-{[(Triisopropylsilyl)oxy]methyl}cyclopentanamine (3.86 g, 14.2 mmol)was added followed by triethylamine (3.30 mL, 23.7 mmol). The mixturewas stirred at room temperature overnight. The reaction mixture wastransferred to a separatory funnel transferring with ethyl acetate (250mL). The organic layer was washed twice with a 10% citric acid (aq) (20mL)/brine (60 mL) mixture. The organic layer was dried over sodiumsulfate, filtered and the solvent removed in vacuo to afford a yellowoil. The product was purified by silica gel chromatography using agradient from 1:9 to 2:3 ethyl acetate:hexanes which afforded 2.48 g(41% yield) of the desired product as a pale yellow oil. NMR (CDCl₃) δ8.58 (s, 1H), 4.72 (m, 1H), 4.46 (m, 1H), 4.16 (q, 2H, J=6.9 Hz), 3.52(m, 1H), 2.96 (m, 2H), 2.54 (s, 3H), 2.31 (m, 3H), 1.81-1.52 (m, 5H),1.24 (t, 3H, J=6.9 Hz) 1.08-0.96 (m, 21H); MS (ESI+) for C₂₆H₄₃N₃O₄SSim/z 522.2 (M+H)⁺.

Ethyl2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Cyclization of ethyl4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and1-{[(triisopropylsilyl)oxy]methyl}cyclohexanamine using GeneralProcedure C afforded the desired product in 17% yield as a pale yellowoil. NMR (CDCl₃) δ 8.63 (s, 1H), 4.83 (m, 1H), 4.73 (m, 1H), 4.14 (q,2H, J=6.0 Hz), 3.86 (m, 1H), 2.97 (m, 2H), 2.55 (s, 3H), 1.97 (m, 1H),1.72-1.48 (m, 9H), 1.23 (t, 3H, J=6.0 Hz), 1.13-0.95 (m, 21H); MS (ESI+)for C₂₇H₄₅N₃O₄SSi m/z 536.2 (M+H)⁺.

Ethyl8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Cyclization of ethyl4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and1-{[tert-butyl(dimethyl)silyl]oxy}-2-methylpropan-2-amine using GeneralProcedure C afforded the desired product in 52% yield as a pale yellowoil. NMR (CDCl₃) δ 8.62 (s, 1H), 4.94 (m, 1H), 4.18 (m, 3H), 3.63 (m,1H), 2.93 (m, 2H), 2.57 (s, 3H), 1.71 (s, 3H), 1.55 (s, 3H), 1.23 (t,3H, J=7.2 Hz), 0.89 (s, 9H), 0.06 (s, 3H), 0.01 (s, 3H); MS (ESI+) forC₂₁H₃₅N₃O₄SSi m/z 454.3 (M+H)⁺.

Ethyl2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Cyclization of ethyl4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and2-[(triisopropylsilyl)oxy]ethanamine using General Procedure C affordedthe desired product in 45% yield as a pale yellow oil. NMR (CDCl₃) δ8.60 (s, 1H), 4.72 (m, 1H), 4.63 (m, 1H), 4.19 (q, 2H, J=6.0 Hz), 3.97(m, 2H), 3.22 (m, 1H), 3.01 (m, 2H), 2.54 (s, 3H), 1.28 (t, 3H, J=6.0Hz), 1.17-1.02 (m, 21H); MS (ESI+) for C₂₂H₃₇N₃O₄SSi m/z 468.1 (M+H)⁺.

tert-Butyl2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate

Cyclization of tert-Butyl4-[4-chloro-2-(methylthio)pyrimidin-5-yl]-4-oxobut-2-enoate and 0.5 Mammonia/dioxane using General Procedure C afforded the desired productin 60% yield as an off-white solid. NMR (CDCl₃) δ 8.66 (s, 1H), 6.18(bs, 1H), 4.34 (m, 1H), 3.05-2.80 (m, 2H), 2.56 (s, 3H), 1.51 (s, 9H);MS (ESI+) for C₁₃H₁₇N₃O₃S m/z 296.0 (M+H)⁺.

General Procedure D

2-(Methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid

Ethyl2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate(2.48 g, 4.75 mmol) was taken up in tetrahydrofuran (10 mL) andacetonitrile (10 mL) at room temperature. 1M Sodium hydroxide (10 mL, 10mmol) was added at room temperature for 1 hour. The reaction wasquenched by the addition of 10% citric acid till pH ca 6-7. The reactionmixture was transferred to a separatory funnel with water (30 mL) andethyl acetate (150 mL). The aqueous layer was removed and the organiclayer washed with brine (50 mL). The organic layer was dried over sodiumsulfate, filtered and the solvent concentrated in vacuo to afford the2.08 g (89% yield) of the desired product as a dark yellow oil. NMR(CDCl₃) δ 8.46 (s, 1H), 4.58 (m, 1H), 4.39 (m, 1H), 3.71 (m, 1H), 2.88(m, 2H), 2.51 (s, 3H), 2.26 (m, 3H), 1.97-1.45 (m, 6H) 1.12-0.92 (m,21H); MS (ESI+) for C₂₄H₃₉N₃O₄SSi m/z 494.2 (M+H)⁺.

2-(Methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid

Saponification of ethyl2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylateusing General Procedure D affords the desired product in 95% yield as adark yellow oil. NMR (CDCl₃) δ 8.58 (s, 1H), 4.75 (m, 1H), 4.53 (m, 1H),3.96 (m, 1H), 2.99 (m, 2H), 2.54 (s, 3H), 1.93-1.48 (m, 10H), 1.13-0.95(m, 21H); MS (ESI+) for C₂₅H₄₁N₃O₄SSi m/z 508.1 (M+H)⁺.

8-(2-{[tert-Butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid

Saponification of ethyl8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylateusing General Procedure D affords the desired product in 99% yield as apale yellow foam. NMR (CDCl₃) δ 8.69 (s, 1H), 4.88 (m, 1H), 4.51 (m,1H), 3.82 (m, 1H), 3.17 (m, 1H), 2.79 (m, 1H), 2.57 (s, 3H), 1.65 (s,3H), 1.60 (s, 3H), 0.92 (s, 9H), 0.11 (s, 6H); MS (ESI+) forC₁₉H₃₁N₃O₄SSi m/z 426.3 (M+H)⁺.

2-(Methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid

Saponification of ethyl2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylateusing General Procedure D affords the desired product in 98% yield as anorange foam. NMR (CDCl₃) δ 8.56 (s, 1H), 4.71 (m, 1H), 4.54 (m, 1H),3.99 (m, 2H), 3.32 (m, 1H), 3.01 (m, 2H), 2.53 (s, 3H), 1.16-0.98 (m,21H); MS (ESI+) for C₂₀H₃₃N₃O₄SSi m/z 440.2 (M+H)⁺.

2-(Methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid

tert-Butyl2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate(220 mg, 0.74 mmol) was taken up in trifluoroacetic acid (5 mL) at roomtemperature under argon. The mixture was stirred at room temperature for45 minutes. The solvent was removed in vacuo to a pink oil which wasre-evaporated first from toluene and finally methanol affording 180 mg(99% yield) of the desired product as an off-white solid. NMR (MeOH-d₄)δ 8.49 (s, 1H), 4.59 (m, 1H), 3.16-2.91 (m, 2H), 2.63 (s, 3H); MS (ESI+)for C₉H₉N₃O₃S m/z 240.0 (M+H)⁺.

General Procedure E

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

2-(Methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid (2.08 g, 4.21 mmol) was taken up in N,N-dimethylformamide (30 mL)at room temperature. 1-(2,4-dimethoxyphenyl)methanamine (1.26 mL, 8.42mmol) was added followed by the addition ofN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (4.80 g, 12.6 mmol) and N,N-diisopropylethylamine(4.40 mL, 25.3 mmol). The reaction mixture was stirred overnight at roomtemperature. The product was diluted with water (50 mL) and poured intoa separatory funnel. The mixture was extracted with twice with ethylacetate (150 mL) and the combined organic layers were thrice washed withhalf-saturated aqueous LiCl (20 mL). The combined organic layers weredried over sodium sulfate, filtered and the solvent removed in vacuo toafford a dark yellow oil. The product was purified by silica gelchromatography using a gradient from 1:4 to 2:3 ethyl acetate:hexaneswhich afforded 2.39 g (88% yield) of the desired product as a brownsticky solid. NMR (CDCl₃) δ 8.58 (s, 1H), 7.07 (m, 1H), 6.62 (m, 1H),6.39 (m, 2H), 4.56 (m, 1H), 4.38 (m, 2H), 4.20 (m, 1H), 3.80 (s, 3H),3.66 (s, 3H), 3.48 (m, 1H), 3.02 (m, 2H), 2.55 (s, 3H), 2.35 (m, 2H),2.06 (m, 1H), 1.70-1.31 (m, 5H), 1.09-0.90 (m, 21H); MS (ESI+) forC₃₃H₅₀N₄O₅SSi m/z 643.2 (M+H)⁺.

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid using General Procedure E afforded the desired product in 52% yieldas a yellow solid. NMR (CDCl₃) δ 8.60 (s, 1H), 6.87 (m, 2H), 6.37 (m,2H), 4.71 (m, 1H), 4.49-4.14 (m, 4H), 3.79 (s, 3H), 3.70 (s, 3H), 3.18(m, 1H), 2.86 (m, 1H), 2.65 (m, 1H), 2.54 (s, 3H), 2.20 (m, 1H), 1.98(m, 2H), 1.61-1.48 (m, 6H), 1.08-0.92 (bs, 21H); MS (ESI+) forC₃₄H₅₂N₄O₅SSi m/z 657.2 (M+H)⁺.

8-(2-{[tert-Butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-N-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid using General Procedure E afforded the desired product in 86% yieldas a viscous yellow oil. NMR (CDCl₃) δ 8.58 (s, 1H), 6.95 (m, 1H), 6.80(m, 1H), 6.37 (m, 2H), 4.71 (m, 1H), 4.27 (m, 2H), 4.13 (m, 1H), 3.84(m, 1H), 3.79 (s, 3H), 3.70 (s, 3H), 3.15 (m, 1H), 2.77 (m, 1H), 2.56(s, 3H), 1.64 (s, 3H), 1.58 (s, 3H), 0.85 (s, 9H), 0.02 (s, 3H), −0.03(s, 3H); MS (ESI+) for C₂₈H₄₂N₄O₅SSi m/z 575.4 (M+H)⁺.

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid using General Procedure E afforded the desired product in 57% yieldas a dark yellow solid. NMR (CDCl₃) δ 8.59 (s, 1H), 7.06 (m, 1H), 6.39(m, 3H), 4.51 (m, 2H), 4.32 (m, 2H), 3.93 (m, 2H), 3.81 (s, 3H), 3.73(s, 3H), 3.18 (m, 1H), 3.01 (m, 2H), 2.54 (s, 3H), 1.11-0.95 (m, 21H);MS (ESI+) for C₂₉H₄₄N₄O₅SSi m/z 589.4 (M+H)⁺.

2-(Methylthio)-5-oxo-N-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Coupling reaction of2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylicacid using General Procedure E afforded the desired product in 94% yieldas a dark yellow oil. NMR (CDCl3) δ 8.64 (s, 1H), 6.09 (bs, 1H), 6.04(bs, 1H), 4.25 (m, 1H), 3.68 (m, 2H), 2.86 (m, 2H), 2.54 (s, 3H),2.07-1.54 (m, 8H), 1.33-0.96 (m, 21H); MS (ESI+) for C₂₄H₄₀N₄O₃SSi m/z493.1 (M+H)⁺.

General Procedure F

N-(2,4-Dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

N-(2,4-Dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide(2.39 g, 3.72 mmol) was taken up in tetrahydrofuran (42 mL) at roomtemperature. Tetra-n-butylammonium fluoride (5.6 mL, 5.6 mmol, 1Msolution in THF) was added and the reaction stirred for 10 minutes atroom temperature. The reaction mixture was concentrated in vacuo to anorange oil and was transferred to a separatory funnel and partitionedbetween ethyl acetate (200 mL) and water (50 mL). The aqueous layer wasremoved and the organic layer washed with water (50 mL) and brine (50mL). The organic layer was dried over sodium sulfate, filtered and thesolvent removed in vacuo to afford a dark yellow semi-solid. The productwas purified by reverse phase chromatography using a gradient from 1:9to 3:2 acetonitrile:water (0.1% TFA). Lyophilization of the desiredfractions afforded 1.81 g (99% yield) of the desired product as a darkyellow powder. NMR (CDCl₃) δ 8.59 (s, 1H), 7.21 (m, 1H), 7.02 (m, 1H),6.39 (m 2H), 4.56 (m, 1H), 4.29 (m, 2H), 3.79 (s, 3H), 3.75 (s, 3H),3.70 (m, 2H), 3.41 (m, 1H), 3.20 (m, 1H), 2.87 (m, 1H), 2.55 (s, 3H),2.18 (m, 1H), 1.97-1.59 (m, 7H); MS (ESI+) for C₂₄H₃₀N₄O₅S m/z 487.1(M+H)⁺.

N-(2,4-Dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclohexyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation ofN-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-8-(1-{[(triisopropylsilyl)oxy]methyl}cyclohexyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure F afforded the desired product in 97% yield as ayellow powder. NMR (CDCl₃) δ 8.69 (s, 1H), 7.58 (m, 1H), 7.07 (m, 1H),6.48 (m, 2H), 4.72 (m, 2H), 4.38 (m, 2H), 3.89 (s, 3H), 3.87 (s, 3H),3.31 (m, 1H), 2.99 (m, 1H), 2.81 (m, 1H), 2.64 (s, 3H), 2.11 (m, 3H),1.94-1.58 (m, 7H); MS (ESI+) for C₂₅H₃₂N₄O₅S m/z 501.1 (M+H)⁺.

8-(2-Hydroxy-1,1-dimethylethyl)-N-(4-methoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation of8-(2-{[tert-butyl(dimethyl)silyl]oxy}-1,1-dimethylethyl)-N-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure F afforded the desired product in 98% yield as ayellow powder. NMR (CDCl₃) δ 8.61 (s, 1H), 7.51 (m, 1H), 7.01 (m, 1H),6.37 (m, 2H), 4.72 (m, 1H), 4.65 (m, 1H), 4.26 (m, 2H), 3.80 (s, 3H),3.76 (s, 3H), 3.63 (m, 1H), 3.18 (m, 1H), 2.80 (m, 1H), 2.57 (s, 3H),1.58 (s, 3H), 1.56 (s, 3H); MS (ESI+) for C₂₂H₂₈N₄O₅S m/z 461.4 (M+H)⁺.

N-(2,4-Dimethoxybenzyl)-8-(2-hydroxyethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation ofN-(2,4-dimethoxybenzyl)-2-(methylthio)-5-oxo-8-{2-[(triisopropylsilyl)oxy]ethyl}-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure F afforded the desired product in 97% yield as apale yellow powder. NMR (CDCl₃) δ 8.59 (s, 1H), 7.02 (m, 1H), 6.69 (m,1H), 6.43 (m, 2H), 4.34 (m, 3H), 3.88 (m, 4H), 3.81 (s, 3H), 3.78 (s,3H), 3.02 (m, 2H), 2.55 (s, 3H); MS (ESI+) for C₂₀H₂₄N₄O₅S m/z 432.9(M+H)⁺.

N-[1-(Hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide

Desilylation of2-(methylthio)-5-oxo-N-(1-{[(triisopropylsilyl)oxy]methyl}cyclopentyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure F afforded the desired product in 31% yield asan off white powder. NMR (CDCl₃) δ 8.66 (s, 1H), 6.19 (bs, 1H), 5.97(bs, 1H), 4.31 (m, 1H), 3.69 (s, 2H), 2.92 (m, 2H), 2.56 (s, 3H),1.95-1.65 (m, 9H); MS (ESI+) for C₁₅H₂₀N₄O₃S m/z 337.0 (M+H)⁺.

General Procedure G

8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

N-(2,4-Dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclopentyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamide(1.81 g, 3.72 mmol) was taken up in methylene chloride (30 mL) at roomtemperature. Triethylamine (1.3 mL, 9.3 mmol) was added followed by therapid addition of methanesulfonyl chloride (0.43 mL, 5.6 mmol). Thereaction mixture was stirred at room temperature for 30 minutes beforebeing heated at reflux overnight. The solvent was removed in vacuoaffording a brown semi-solid. The product was purified by reverse phasechromatography using a gradient from 1:9 to 3:2 acetonitrile:water (0.1%TFA). Lyophilization of the desired fractions gave 764 mg (44% yield) ofthe desired product as a light brown powder. NMR (CDCl₃) δ 8.49 (s, 1H),7.13 (m, 1H), 6.43 (m, 2H), 5.49 (m, 1H), 4.37-4.16 (m, 4H), 3.81 (s,3H), 3.80 (s, 3H), 3.29 (m, 1H), 2.95 (m, 1H), 2.82 (s, 3H), 2.41 (m,1H), 1.99-1.52 (m, 7H); MS (ESI+) for C₂₄H₂₈N₄O₄S m/z 469.1 (M+H)⁺.

8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

Mesylation and cyclization ofN-(2,4-dimethoxybenzyl)-8-[1-(hydroxymethyl)cyclohexyl]-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure G afforded the desired product in 73% yield as awhite powder. NMR (MeOH-d₄) δ 8.48 (s, 1H), 7.14 (m, 1H), 6.43 (m, 1H),6.39 (m, 1H), 5.61 (m, 1H), 4.31 (m, 1H), 4.21 (m, 2H), 3.83 (s, 3H),3.80 (s, 3H), 3.38 (m, 1H), 3.22 (m, 1H), 2.95 (m, 1H), 2.82 (s, 3H),2.22 (m, 1H), 1.99-1.09 (m, 9H); MS (ESI+) for C₂₅H₃₀N₄O₄S m/z 483.1(M+H)⁺.

8-(2,4-Dimethoxybenzyl)-10,10-dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Mesylation and cyclization of8-(2-Hydroxy-1,1-dimethylethyl)-N-(4-methoxybenzyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure G afforded the desired product in 82% yield as ayellow powder. NMR (CDCl₃) δ 8.45 (s, 1H), 7.14 (m, 1H), 6.40 (m, 2H),5.56 (m, 1H), 4.40 (m, 1H), 4.31 (m, 2H), 4.13 (m, 1H), 3.81 (s, 3H),3.79 (s, 3H), 3.28 (m, 1H), 2.88 (m, 1H), 2.79 (s, 3H), 1.80 (s, 3H),1.45 (s, 3H); MS (ESI+) for C₂₂H₂₆N₄O₄S m/z 443.5 (M+H)⁺.

8-(2,4-Dimethoxybenzyl)-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Mesylation and cyclization ofN-(2,4-dimethoxybenzyl)-8-(2-hydroxyethyl)-2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxamideusing General Procedure G afforded the desired product in 81% yield as alight brown powder. NMR (CDCl₃) δ 8.52 (s, 1H), 7.11 (m, 1H), 6.44 (m,2H), 5.31 (m, 1H), 4.68-4.29 (m, 5H), 4.04 (m, 1H), 3.81 (s, 3H), 3.80(s, 3H), 3.20 (m, 1H), 3.01 (m, 1H), 2.82 (s, 3H); MS (ESI+) forC₂₀H₂₂N₄O₄S m/z 415.0 (M+H)⁺.

General Procedure H

2′-(Methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione(764 mg, 1.63 mmol) was taken up in trifluoroacetic acid (10 mL) at roomtemperature under argon. The mixture was heated to 75° C. for 6 hours,cooled to room temperature and left to stir overnight. The solvent wasremoved in vacuo to afford a purple oil. The product was purified byreverse phase chromatography using a gradient from 100% water (0.1% TFA)to 1:1 acetonitrile:water (0.1% TFA). Lyophilization of the desiredfractions afforded 117 mg (23% yield) of the desired product as a paleyellow powder. NMR (CDCl₃) δ 8.52 (s, 1H), 5.58 (m, 2H), 4.34 (bs 2H),3.29 (m, 1H), 3.06 (m, 1H), 2.84 (s, 3H), 2.48 (m, 1H), 2.31 (m, 1H),2.11-1.65 (m, 6H); MS (ESI+) for C₁₅H₁₈N₄O₂S m/z 319.0 (M+H)⁺.

2′-(Methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

Removal of the dimethoxybenzyl group of8′-(2,4-Dimethoxybenzyl)-2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dioneusing General Procedure H afforded the desired product in 23% yield as awhite powder. NMR (MeOH-d₄) δ 8.60 (s, 1H), 4.78 (m, 1H), 4.54 (m, 2H),3.38 (m, 1H), 2.86 (s, 3H), 2.84 (m, 1H), 2.12-1.30 (m, 10H); MS (ESI+)for C₁₆H₂₀N₄O₂S m/z 333.1 (M+H)⁺.

10,10-Dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Removal of the dimethoxybenzyl group of8-(2,4-dimethoxybenzyl)-10,10-dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dioneusing General Procedure H afforded the desired product in 56% yield as awhite powder. NMR (CDCl₃) δ 8.54 (s, 1H), 5.65 (m, 1H), 5.51 (bs, 1H),4.31 (s, 2H), 3.23 (m, 1H), 3.04 (m, 1H), 2.85 (s, 3H), 1.78 (s, 3H),1.68 (s, 3H); MS (ESI+) for C₁₃H₁₆N₄O₂S m/z 293.2 (M+H)⁺.

2-(Methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

Removal of the dimethoxybenzyl group of8-(2,4-dimethoxybenzyl)-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dioneusing General Procedure H afforded the desired product in 21% yield as abrown powder. NMR (MeOH-d₄) δ 8.65 (s, 1H), 4.78-4.05 (m, 5H), 3.32 (m,2H), 3.01 (m, 1H), 2.87 (s, 3H); MS (ESI+) for C₁₁H₁₂N₄O₂ m/z 265.0(M+H)⁺.

General Procedure I

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

2′-(Methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclopentane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione(117 mg, 0.367 mmol) was taken up in N,N-dimethylacetamide (4.0 mL, 43mmol) at room temperature under argon.5-(4-methylpiperazin-1-yl)pyridin-2-amine (100 mg, 0.55 mmol) was addedand the reaction mixture was heated just to 150° C. and then immediatelyremoved from the heat and cooled to room temperature. The product waspurified by reverse phase chromatography using a gradient from 100%Water (0.1% TFA) to 1:1 acetonitrile:water (0.1% TFA). Lyophilization ofthe desired fractions afforded 11 mg (7% yield) of the desired productas an orange powder. NMR (MeOH-d₄) δ 8.54 (s, 1H); 8.06 (m, 1H), 7.85(m, 1H), 7.69 (m, 1H), 4.69 (m, 1H), 4.39 (m, 2H), 3.95 (m, 2H), 3.69(m, 2H), 3.39-3.15 (m, 5H), 3.02 (s, 3H), 2.77 (m, 1H), 2.21 (m, 1H),2.09-1.67 (m, 7H); MS (ESI+) for C₂₄H₃₀N₈O₂ m/z 463.1 (M+H)⁺.

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione

S_(N)Ar reaction using General Procedure I and2′-(methylthio)-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dioneafforded the desired product in 38% yield as a yellow powder. NMR(MeOH-d₄) δ 8.52 (s, 1H), 8.07 (m, 1H), 7.89 (bs, 1H), 7.71 (m, 1H),4.71 (m, 1H), 4.40 (m, 2H), 3.94 (m, 2H), 3.68 (m, 2H), 3.35-3.22 (m,5H), 3.02 (s, 3H), 2.73 (m, 1H), 2.02-1.25 (m, 10H); MS (ESI+) forC₂₅H₃₂N₈O₂ m/z 477.2 (M+H)⁺.

10,10-Dimethyl-2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

S_(N)Ar reaction using General Procedure I and10,10-dimethyl-2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dioneafforded the desired product in 10% yield as a yellow powder. NMR(MeOH-d₄) δ 8.52 (s, 1H), 8.05 (m, 2H), 7.86 (m, 1H), 7.67 (m, 1H), 4.66(m, 1H), 4.33 (m, 2H), 3.93 (m, 2H), 3.68 (m, 2H), 3.38-3.21 (m, 5H),3.01 (s, 3H), 2.72 (m, 1H), 1.65 (s, 3H), 1.54 (s, 3H); MS (ESI+) forC₂₂H₂₈N₈O₂ m/z 437.4 (M+H)⁺.

2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dione

S_(N)Ar reaction using General Procedure I and2-(methylthio)-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dioneafforded the desired product in 15% yield as an orange powder. NMR(DMSO-d₆) δ 8.41 (s, 1H), 8.05 (m, 1H), 7.93 (m, 1H), 7.82 (m, 1H),4.59-4.34 (m, 3H), 4.03-3.84 (m, 4H), 3.49 (m, 2H), 3.30-3.09 (m, 6H),2.80 (s, 3H), 2.80-2.67 (m, 2H); MS (ESI+) for C₂₀H₂₄N₈O₂ m/z 409.1(M+H)⁺.

General Procedure J

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydro-7′H-dispiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5′,2″-[1,3]dithian]-7′-one

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine]-5′,7′-dione(90.0 mg, 0.189 mmol) and 1,3-propanedithiol (0.0379 mL, 0.378 mmol)were taken up in toluene (5 mL) at room temperature under argon.p-Toluenesulfonic acid (0.02 g, 0.1 mmol) was then added. The reactionvessel was fitted with a condenser and the reaction mixture heated atreflux overnight. The reaction mixture was cooled to room temperatureand the solvent removed in vacuo affording a thick dark yellow oil. Theproduct was purified by reverse phase chromatography using a gradientfrom 100% water (0.1% TFA) to 3:2 acetonitrile:water (0.1% TFA).Lyophilization of the desired fractions afforded 35 mg (33% yield) ofthe desired product as a pale yellow powder. NMR (MeOH-d₄) δ 8.52 (s,1H), 7.90 (m, 1H), 7.84 (m, 1H), 7.52 (m, 1H), 4.64 (m, 1H), 4.53 (m,1H), 4.16 (m, 1H), 3.60 (m, 2H), 3.41-3.26 (m, 6H), 3.01 (s, 3H), 2.91(m, 1H), 2.75 (m, 1H), 2.61 (m, 1H), 2.21 (m, 1H), 2.11 (m, 1H),1.95-1.72 (m, 10H), 1.61 (m, 1H), 1.33 (m 2H); MS (ESI+) for C₂₈H₃₈N₈OS₂m/z 567.1 (M+H)⁺.

10′,10′-Dimethyl-2′-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,9′,10′-tetrahydrospiro[1,3-dithiane-2,5′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidin]-7′(8′H)-one

Dithiane formation using General Procedure J and10,10-dimethyl-2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5,7(8H)-dioneafforded the desired product in 43% yield as an orange powder. NMR(MeOH-d₄) δ 8.53 (s, 1H), 7.88 (m, 1H), 7.82 (m, 1H), 7.47 (m, 1H), 4.47(m, 1H), 4.41 (m, 1H), 4.16 (m, 1H), 3.92-3.15 (m, 11H), 3.00 (s, 3H),2.90-2.81 (m, 3H), 2.21 (m, 1H), 1.87 (m, 1H), 1.60 (s, 3H), 1.48 (s,3H); MS (ESI+) for C₂₅H₃₄N₈OS₂ m/z 527.1 (M+H)⁺.

General Procedure K

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydrospiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidin]-7′(5′H)-one

2′-{[5-(4-Methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,8′,9′-tetrahydro-7′H-dispiro[cyclohexane-1,10′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidine-5′,2″-[1,3]dithian]-7′-one(35 mg, 0.062 mmol) in ethanol (1 ml) was added to Raney nickel (1 mL ofthe aqueous slurry which was washed thrice with ethanol decanting offthe ethanol after each washing) in ethanol (3 mL) under argon. Thereaction mixture was heated to 45° C. for 30 minutes. After cooling toroom temperature, the reaction mixture was filtered through a pad ofCelite® washing with ethanol. The solvent was removed in vacuo affordinga yellow oil. The product was purified by reverse phase chromatographyusing a gradient from 100% water (0.1% TFA) to 3:2 acetonitrile:water(0.1% TFA). Lyophilization of the desired fractions afforded 4 mg (14%yield) of the desired product as a pale yellow powder. NMR (CDCl₃) δ7.95 (m, 1H), 7.93 (s, 1H), 7.77 (m, 1H), 7.51 (m, 1H), 4.57 (m, 1H),4.51 (m, 1H), 4.35 (m, 1H), 3.89 (m, 2H), 3.69 (m, 2H), 3.37 (m, 2H),3.15 (m, 2H), 3.01 (s, 3H), 2.79 (m, 1H), 2.58 (m, 1H), 2.39 (m, 1H),2.05-1.45 (m, 11H); MS (ESI+) for C₂₅H₃₄N₈O m/z 463.1 (M+H)⁺.

10,10-Dimethyl-2-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6,6a,9,10-tetrahydro-5H-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidin-7(8H)-one

Desulfurization using General Procedure K and10′,10′-dimethyl-2′-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-6′,6a′,9′,10′-tetrahydrospiro[1,3-dithiane-2,5′-pyrazino[1′,2′:1,6]pyrido[2,3-d]pyrimidin]-7′(8′H)-oneafforded the desired product in 11% yield as a yellow powder. NMR(MeOH-d₄) δ 7.93 (m, 2H), 7.75 (m, 1H), 7.43 (m, 1H), 4.52 (m, 1H), 4.32(m, 2H), 3.89 (m, 2H), 3.67 (m, 2H), 3.38 (m, 2H), 3.14 (m, 2H), 3.01(s, 3H), 2.79 (m, 1H), 2.60 (m, 1H), 2.39 (m, 1H), 2.00 (m, 1H), 1.55(s, 3H), 1.54 (s, 3H); MS (ESI+) for C₂₂H₃₀N₈O m/z 423.1 (M+H)⁺.

General Procedure L

(1-Aminocyclohexyl)methanol

2M Lithium tetrahydroaluminate in tetrahydrofuran (80.0 mL, 160 mmol)was charged into a 500 mL 3-necked round bottomed flask (oven-dried andcooled under argon) fitted with a magnetic stir bar and the solution wascooled to 0° C. under argon. 1-Aminocyclohexanecarboxylic acid (7.64 g,53.3 mmol) is added portionwise over a period of 1 hour. At the end ofthe addition, the reaction mixture was diluted with tetrahydrofuran (60mL), slowly warmed to room temperature, and then heated at reflux for 18hours. The mixture was cooled to room temperature. The reaction mixturewas further diluted with tetrahydrofuran (160 mL) and then cooled to 0°C. Saturated aqueous sodium carbonate (100 ml) was added very slowlykeeping the internal temperature below 15° C. After the addition of thecarbonate solution is complete, the ice bath was left to expire and themixture slowly warmed to room temperature overnight. The reactionmixture was filtered thru a pad of Celite® washing with ethyl acetate(400 mL). The solvent was removed in vacuo to afford a wet oil which wastaken up in methylene chloride (300 mL) and dried over sodium sulfate.Filtration and concentration of the solvent in vacuo affords 6.89 g (99%yield) of the desired product as a clear colorless oil. NMR (CDCl₃) 3.34(s, 2H), 1.81 (bs, 3H), 1.51-1.32 (m, 10H); MS (ESI+) for C₇H₁₅NO m/z130.0 (M+H)⁺.

(1-Aminocyclopentyl)methanol

Using General Procedure L on commercially available cycloleucine affordsthe desired product in 99% yield as a pale yellow oil. NMR (CDCl₃) 3.40(s, 2H), 1.86-1.61 (m, 9H), 1.46-1.29 (m, 2H); MS (ESI+) for C₆H₁₃NO m/z116.1 (M+H)⁺.

General Procedure M

1-{[(Triisopropylsilyl)oxy]methyl}cyclohexanamine

(1-Aminocyclohexyl)methanol (3.43 g, 26.5 mmol) was taken up inmethylene chloride (80 mL) at room temperature under argon.Triethylamine (5.6 mL, 40 mmol) was added followed by the addition oftriisopropylsilyl chloride (5.34 mL, 25.2 mmol). The reaction mixturewas stirred at room temperature overnight during which time it becameturbid. The reaction mixture was poured into a separatory funneltransferring with methylene chloride (100 mL). The organic layer waswashed sequentially with water (40 mL×2) and brine (40 mL). The organiclayer was dried over sodium sulfate, filtered and the solventconcentrated in vacuo to afford 6.68 g (93% yield) of the desiredproduct as a clear pale yellow oil. NMR (CDCl₃) δ 3.49 (s, 2H),1.75-1.25 (m, 10H), 1.16-1.06 (m, 21H); MS (ESI+) for C₁₁H₂₇NOSi m/z203.2 (M+H)⁺.

1-{[(Triisopropylsilyl)oxy]methyl}cyclopentanamine

Following General Procedure M and using (1-aminocyclopentyl)methanol thedesired product was obtained in 85% yield as a clear dark yellow oil.NMR (CDCl₃) δ 3.53 (s, 2H), 1.85-1.39 (m, 8H), 1.16-1.07 (m, 21H); MS(ESI+) for C₁₅H₃₃NOSi m/z 272.2 (M+H)⁺.

2-[(Triisopropylsilyl)oxy]ethanamine

Following General Procedure M and using commercially availableethanolamine the desired product was obtained in 99% yield as a clearpale yellow oil. NMR (CDCl₃) δ 3.56 (t, 2H, J=6.0 Hz), 2.94 (t, 2H,J=6.0 Hz), 1.09-0.99 (m, 21H); MS (ESI+) for C₁₁H₂₇NOSi m/z 217.2(M+H)⁺.

1-{[tert-Butyl(dimethyl)silyl]oxy}-2-methylpropan-2-amine

Following General Procedure M and using commercially available2-amino-2-methyl-1-propanol and using tert-butyldimethylsilyl chloridethe desired product was obtained in 95% yield as a clear colorless oil.NMR (CDCl₃) δ 3.31 (s, 2H), 0.93 (s, 9H), 0.06 (s, 6H); MS (ESI+) forC₁₀H₂₅NOSi m/z 204.2 (M+H)⁺.

As exemplified in Scheme 10, compounds of Formula VI can be synthesizedbeginning with the aldehyde illustrated above. In Step 1, an alkyne canbe treated with an organic solvent, and a base optionally at a reducedtemperature and subsequently treated with an aldehyde according tomethods known in the art. For example, the aldehyde in Step 1 can betreated with a base, for example, isopropylmagnesium chloride lithiumchloride complex in an organic solvent, for example, tetrahydrofuran atabout −15° C. and next treated with an aldehyde to generate an alkyne.In Step 2, a desired alkynyl alcohol can be treated with a base in anorganic solvent at an elevated temperature to isomerize the desiredalkynyl alcohol to a desired alkene. For example, a desired alkynylalcohol can be treated with a base, such as triethylamine in an organicsolvent, for example, 1,4-dioxane at an elevated temperature of about60° C. to generate an alkene. In Step 3, a desired alkene can be treatedwith ammonia and a mixture of organic solvents to form a tert-butyl2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylateaccording to methods known in the art. For example, an alkene can betreated with 0.5M ammonia and a mixture of organic solvents, forexample, dioxane and acetonitrile to form a tert-butyl2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate.In Step 4, a desired ester can be treated with a desired acid and anorganic solvent to generate a desired carboxylic acid according tomethods known in the art. For example, a desired ester can be treatedwith a desired acid, for example, trifluoroacetic acid, to generate acarboxylic acid. In one embodiment, the organic solvent isdichloromethane. In Step 5, a desired acid can be treated with a desiredamine, an organic solvent and a coupling reagent to form a desired amideaccording to methods known in the art. For example, a desired acid canbe treated with a desired amine, an organic solvent, for example,N,N-dimethylformamide, and a coupling reagent, for example,1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, to generate an amide. In Step 6, a silylprotected alcohol can be treated with a fluoride reagent and an organicsolvent according to methods known in the art to generate a desiredalcohol. For example, a silyl protected alcohol can be treated with afluoride reagent, for example, tetrabutylammonium fluoride, and anorganic solvent, for example, acetonitrile, to generate an alcohol. Instep 7, a desired alcohol can be treated with a sulfonyl chloride togenerate a desired mesylate according to methods known in the art. Forexample, an alcohol can be treated with a desired sulfonyl chloride, forexample, methanesulfonyl chloride, to generate a mesylate. In oneembodiment, an amine spontaneously reacts with said mesylate to generatea cyclic amide. In Step 8, a desired thiol can be treated with a desiredamine and an organic solvent at an elevated temperature to generate adesired amine according to methods known in the art. For example, athiol can be treated with an amine, for example,5-(4-methylpiperazin-1-yl)pyridin-2-amine, and an organic solvent, forexample, N,N-dimethylacetamide, at an elevated temperature of about 150°C. to generate an amine. The compound5-(4-methylpiperazin-1-yl)pyridin-2-amine can be prepared as disclosedin U.S. Pat. No. 8,598,186 to Tavares and Strum.

As exemplified in Scheme 11, compounds of Formula VI can be synthesizedbeginning with the aldehyde illustrated above. In Step 1, an alkyne canbe treated with an organic solvent, and a base optionally at a reducedtemperature and subsequently treated with an aldehyde according tomethods known in the art. For example, the aldehyde in Step 1 can betreated with a base, for example, isopropylmagnesium chloride lithiumchloride complex in an organic solvent, for example, tetrahydrofuran atabout −15° C. and next treated with an aldehyde to generate an alkyne.In Step 2, a desired alkynyl alcohol can be treated with a base in anorganic solvent at an elevated temperature to isomerize the desiredalkynyl alcohol to a desired alkene. For example, a desired alkynylalcohol can be treated with a base, such as triethylamine in an organicsolvent, for example, 1,4-dioxane at an elevated temperature of about60° C. to generate an alkene. In Step 3, a desired alkene can be treatedwith ammonia and a mixture of organic solvents to form a tert-butyl2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylateaccording to methods known in the art. For example, an alkene can betreated with 0.5M ammonia and a mixture of organic solvents, forexample, dioxane and acetonitrile to form a tert-butyl2-(methylthio)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-7-carboxylate.In Step 4, an amine can be treated with a base, an organic solvent, anda cyclic sulfamidate to form an amine according to methods known in theart. For example, a desired amine can be treated with a base, forexample, triethylamine, an organic solvent, for example,N,N-dimethylformamide, and a cyclic sufamidate to form an amine. In Step5, a protected amine can be treated with an organic acid to form anamine that can subsequently form a cyclic amide according to methodsknown in the art. For example, a protected amine can be treated with anorganic acid, for example, trifluoroacetic acid, and subsequently reactwith an ester to form a cyclic amide.

EXAMPLES

The patents WO 2013/148748 entitled “Lactam Kinase Inhibitors” toTavares, F. X., WO 2013/163239 entitled “Synthesis of Lactams” toTavares, F. X., and U.S. Pat. No. 8,598,186 entitled “CDK Inhibitors” toTavares, F. X. and Strum, J. C. are incorporated by reference herein intheir entirety.

Example 1 Synthesis of Compound 2 (Scheme 1)

Compound 2 is synthesized according to the method of A. Haidle et al.,See, WO 2009/152027 entitled5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives for MARKinhibition.

Example 2 Synthesis of Compound 3 (Scheme 1)

Step 1: A round-bottomed flask inerted with a nitrogen atmosphere ischarged with Compound 2, ethanol, and lithium borohydride at ambienttemperature. The reaction is stirred at ambient temperature andmonitored by thin layer chromatography (TLC) or HPLC. Once Compound 2can no longer be detected, the reaction is quenched with an aqueous acidsuch as aqueous hydrochloric acid, diluted with ethyl acetate and thelayers separated. The organic layer is dried over anhydrous magnesiumsulfate, filtered and concentrated in vacuo. The product, a primaryalcohol, is purified by silica gel column chromatography eluting with ahexane-ethyl acetate gradient and used directly in the next step.

Step 2: A round-bottomed flask inerted with a nitrogen atmosphere ischarged with the primary alcohol prepared in step 1, DMF and phosphorustribromide. The reaction is stirred at ambient temperature and monitoredby thin layer chromatography (TLC) or HPLC. Once the primary alcohol canno longer be detected, the reaction is quenched with brine and dilutedwith toluene. The layers are separated and the toluene layer is driedover anhydrous magnesium sulfate, filtered and concentrated in vacuo.The bromide is purified by silica gel column chromatography eluting witha hexane-ethyl acetate gradient.

Example 3 Synthesis of Compound 5 (Scheme 1)

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith tetrahydrofuran and the lactam 4, described below. The reaction iscooled to −78° C. and lithium diisopropylamide solution (2M inTHF/heptane/ethyl benzene) is added dropwise. To the resulting enolateis added Compound 3, dropwise, and the reaction is allowed to warm toroom temperature overnight. The reaction is diluted with saturated brineand the layers are separated. The organic layer is dried over anhydrousmagnesium sulfate, filtered and concentrated in vacuo. The product ispurified by silica gel column chromatography eluting with adichloromethane-methanol gradient.

Example 4 Synthesis of Compound 6 (Scheme 1)

A round-bottomed flask is charged with Compound 5 and an aqueous acid,for example a pH=1 HCl solution. The reaction is allowed to stir at roomtemperature until starting material is no longer detected by thin layerchromatography or HPLC. The reaction is neutralized with solid K₂CO₃ anddiluted with dichloromethane. The layers are separated, the organiclayer dried over anhydrous magnesium sulfate, filtered and concentrated.Compound 6 is purified by silica gel column chromatography eluting witha dichloromethane-methanol gradient.

Example 5 Synthesis of Compound 7 (Scheme 1)

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith Compound 6, ethanol and DBU (10 eq). The reaction is monitored bythin layer chromatography or HPLC. Note: The reaction can be heated atreflux if necessary. Once Compound 6 is no longer detected, the reactionis concentrated in vacuo. The lactam 7 is purified by silica gel columnchromatography eluting with a dichloromethane-methanol gradient.

Example 6 Synthesis of Compound 8 (Scheme 1)

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith Compound 7, meta-chloroperoxybenzoic acid, an organic solvent andstirred at ambient temperature. The reaction is monitored by thin layerchromatography or HPLC. Once Compound 7 is no longer detected, thereaction is concentrated in vacuo. Compound 8 is purified by silica gelcolumn chromatography eluting with a dichloromethane-methanol gradient.

Example 7 Synthesis of Compound 10 (Scheme 1)

The tricyclic lactam 8 is combined with an amine (9, 0.9 eq) and anorganic solvent such as tetrahydrofuran. A strong base such as lithiumhexamethyldisilazane is added and the reaction is stirred until lactam 8is no longer detected by either thin layer chromatography or HPLC. Thereaction is concentrated in vacuo. The product is purified by silica gelcolumn chromatography eluting with a dichloromethane-methanol gradient.

Alternatively, a CEM Discovery microwave vessel is charged with thetricyclic lactam 8, N-methyl-2-pyrrolidone (NMP), Hunig's base, andamine 9 (0.9eq). The reaction is heated at 150° C. for 1-4 hours whilebeing monitored by TLC. Once the tricyclic lactam 8 is no longerdetected by TLC or HPLC, the reaction is concentrated in vacuo. Theproduct is purified by silica gel column chromatography eluting with adichloromethane-methanol gradient.

Example 8 Synthesis of Compound 11 (Scheme 2)

Compound 7 is treated with an oxidizing agent such as2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in an organic solvent togenerate the alkene intermediate 12.

Example 9 Synthesis of Compound 14 (Scheme 2)

The sulfone intermediate 12 is combined with an amine (13, 0.9 eq) in anorganic solvent such as tetrahydrofuran. An organic base such as lithiumhexamethyldisilazane is added and the reaction is stirred until sulfoneintermediate 12 can no longer be detected by thin layer chromatographyor HPLC. The product is purified by silica gel column chromatographyeluting with a dichloromethane-methanol gradient.

Alternatively, a CEM Discovery microwave vessel is charged with thesulfone intermediate 12, N-methyl-2-pyrrolidone (NMP), Hunig's base, andamine 13 (0.9eq). The reaction is heated at 150° C. for 1-4 hours whilebeing monitored by TLC. Once the sulfone intermediate 12 is no longerdetected by TLC or HPLC, the reaction is concentrated in vacuo. Theproduct is purified by silica gel column chromatography eluting with adichloromethane-methanol gradient.

Example 10 Synthesis of Compound 4

Step 1: Synthesis of Compound 15 (Scheme 3)

Compound 15 is synthesized according to the method of Arigon, J., See,US 2013/0289031, entitled “Pyrimidinone derivatives, preparation thereofand pharmaceutical use thereof.”

Step 2: Synthesis of Compound 16 (Scheme 3)

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith Compound 15, dichloromethane and triethylamine (1.5 eq). Thereaction is cooled to 0° C. and Boc anhydride (1.5 eq) is added. Thereaction is allowed to stir at room temperature until Compound 15 is nolonger detected by thin layer chromatography or HPLC. The reaction isconcentrated in vacuo. The product is purified by silica gel columnchromatography eluting with a hexane-ethyl acetate gradient.

Step 3: Synthesis of Compound 17 (Scheme 3)

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith Compound 16, acetonitrile and a base such as potassium carbonate.Methyl chloroacetate is added dropwise. The reaction is allowed to stirat room temperature until Compound 16 is no longer detected by thinlayer chromatography or HPLC. The reaction is concentrated in vacuo. Theproduct is purified by silica gel column chromatography eluting with ahexane-ethyl acetate gradient.

Step 4: Synthesis of Compound 18 (Scheme 3)

Compound 17 is dissolved in a solution comprising 3M HCl in methanol andthe reaction is stirred at ambient temperature. Note: the reaction canbe heated at a temperature of about 25° C. to about 60° C. to acceleratethe reaction rate. Once the starting material is no longer detected bythin layer chromatography, the reaction is concentrated in vacuo. Theproduct is purified by silica gel column chromatography using adichloromethane-methanol gradient

Step 5: Synthesis of Compound 19 (Scheme 3)

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith Compound 18, dichloromethane, and diisopropylethylamine (1.2 eq).Chloromethyl methyl ether (MOM-Cl, 1.2 eq) is added dropwise. Thereaction is allowed to stir at room temperature and monitored by TLC.Once the starting material is no longer detected by thin layerchromatography, the reaction is quenched with saturated brine solution.The organic layer is separated, dried over anhydrous magnesium sulfate,filtered and concentrated in vacuo. The product is purified by silicagel column chromatography using a dichloromethane-methanol gradient.

Step 6: Synthesis of Compound 4

A round-bottomed flask inerted with a nitrogen atmosphere is chargedwith anhydrous tetrahydrofuran and Compound 19. The reaction is cooledto −78° C. Sodium bis(trimethylsilyl)amide (1M in THF, 1.1 eq) is addeddropwise. Chloromethyl methyl ether (MOM-Cl, 1.2 eq) is added dropwisewith stirring and the reaction is allowed to warm to room temperatureovernight. The reaction is quenched with saturated brine solution andthe layers are separated. The organic layer is dried over anhydrousmagnesium sulfate, filtered and concentrated in vacuo. The product ispurified by silica gel column chromatography using adichloromethane-methanol gradient.

Example 11 Synthesis of Compound 25 (Scheme 5)

Compound 20 is commercially available. Compound 25 is synthesizedaccording to the synthetic methodology disclosed in Example 10.

Example 12 Synthesis of Compound 31 (Scheme 6)

Compound 31 is synthesized according to the synthetic methodologydisclosed in Example 10.

Example 13 Synthesis of Compound 33 (Scheme 7)

Step 1: Synthesis of Compound 32

Compound 32, 5-morpholinopyrid-2-amine, is synthesized according toTavares, F. X. and Strum, J. C., See, U.S. Pat. No. 8,598,186, entitled“CDK Inhibitors”.

Step 2: Synthesis of Compound 33

The sulfone intermediate 8 is diluted with a suitable solvent such astetrahydrofuran and an organic base such as lithium hexamethyldisilazaneis added. The amine 32 is added and the reaction is stirred untilsulfone intermediate 8 can no longer be detected by thin layerchromatography or HPLC. The product is purified by silica gel columnchromatography eluting with a dichloromethane-methanol gradient.

Alternatively, a CEM Discovery microwave vessel is charged with thesulfone intermediate 8, N-methyl-2-pyrrolidone (NMP), Hunig's base, and5-morpholinopyrid-2-amine (0.9eq). The reaction is heated at 150° C. for1-4 hours while being monitored by TLC. Once the sulfone intermediate 8is no longer detected by TLC or HPLC, the reaction is concentrated invacuo. The product is purified by silica gel column chromatographyeluting with a dichloromethane-methanol gradient.

Example 14 Synthesis of Compound 34 (Scheme 8)

Step 1: Synthesis of Compound 32

Compound 32, 5-morpholinopyrid-2-amine, is synthesized according toTavares, F. X. and Strum, J. C., See, U.S. Pat. No. 8,598,186, entitled“CDK Inhibitors”.

Step 2: Synthesis of Compound 34

The sulfone intermediate 12 is combined with a suitable solvent such astetrahydrofuran and an organic base such as lithiumhexamethyldisilazane. The amine 32 is added and the reaction is stirreduntil sulfone intermediate 12 can no longer be detected by thin layerchromatography or HPLC. The product is purified by silica gel columnchromatography eluting with a dichloromethane-methanol gradient.

Alternatively, a CEM Discovery microwave vessel is charged with thesulfone intermediate 12, N-methyl-2-pyrrolidone (NMP), Hunig's base, and5-morpholinopyrid-2-amine (0.9eq). The reaction is heated at 150° C. for1-4 hours while being monitored by TLC. Once the sulfone intermediate 12is no longer detected by TLC or HPLC, the reaction is concentrated invacuo. The product is purified by silica gel column chromatographyeluting with a dichloromethane-methanol gradient.

Example 15 Preparation of a Formula V compound

Step 1: Compound 7 is Boc protected according to the method of A. Sarkaret al. (JOC, 2011, 76, 7132-7140).

Step 2: Boc-protected Compound 7 is treated with 5 mol % NiCl₂(Ph₃)₂,0.1 eq triphenylphosphine, 3 eq Mn, 0.1 eq tetraethylammonium iodide, inDMI under CO₂ (1 atm) at 25° C. for 20 hours to convert the methyl thiolderivative into the carboxylic acid.

Step 3: The carboxylic acid from Step 2 is converted to thecorresponding acid chloride using standard conditions.

Step 4: The acid chloride from Step 3 is reacted with N-methylpiperazine to generate the corresponding amide.

Step 5: The amide from Step 4 is deprotected using trifluoroacetic acidin methylene chloride to generate the target compound. The product ispurified by silica gel column chromatography eluting with adichloromethane-methanol gradient.

Each of Compounds 33 through 34 and corresponding compounds with variousR⁸, R¹ and Z definitions may be reacted with sodium hydride and an alkylhalide or other halide to insert the desired R substitution prior toreaction with an amine, such as described above for the synthesis ofCompound 33, to produce the desired product of Formulae I, II, III, IV,V, or VI.

Example 16 CDK4/6 Inhibition In Vitro Assay

Selected compounds disclosed herein were tested in CDK4/cyclinD1,CDK6/CycD3, CDK2/CycA, CDK2/cyclinE, CDK5/p25, CDK5/p35, CDK7/CycH/MAT1,and CDK9/CycT kinase assays by Nanosyn (Santa Clara, Calif.) todetermine their inhibitory effect on these CDKs. The assays wereperformed using microfluidic kinase detection technology (Caliper AssayPlatform). The compounds were tested in 12-point dose-response format insinglicate at Km for ATP. Phosphoacceptor substrate peptideconcentration used was 1.25 μM for all assays (except μM 10 was used forthe CKD7/CycH/MAT1 assay and Staurosporine was used as the referencecompound for all assays. Specifics of each assay are as described below:

CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM;Incubation time: 3 hr.

CDK2/CyclinE: Enzyme concentration: 0.2 nM; ATP concentration: 100 μM;Incubation time: 3 hr.

CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM;Incubation time: 3 hr.

CDK6/CyclinD3: Enzyme concentration: 10 nM; ATP concentration: 300 μM;Incubation time: 3 hr.

CDK5/p25: Enzyme concentration: 0.1 nM; ATP concentration: 20 μM;Incubation time: 3 hr.

CDK5/p35: Enzyme concentration: 0.07 nM; ATP concentration: 20 μM;Incubation time: 3 hr.

CDK7/CycH/MAT1: Enzyme concentration: 5 nM; ATP concentration: 50 μM;Incubation time: 3 hr.

CDK9/CycT: Enzyme concentration: 5 nM; ATP concentration: 10 μM;Incubation time: 17 hr.

TABLE 2 Inhibition of CDK kinases by Tricyclic Lactam Compounds CompoundCdk2/ Cdk2/ Cdk4/ Cdk5/ Cdk5/ Cdk6/ Cdk7/CycH/ Cdk9/ No. CycA CycE CycD1p25 p35 CycD3 MAT1 Cyc T ZZZ * * * * * * * YYY * * *** * * ** * BBBB** * ** * * ** * * AAAA * * * * * * * * CCCC * * * * * * * * GGGG * *** * * ** * * * >100 μM ** 10 μM < X > 100 μM *** <10 μM

Example 17 G1 Arrest (Cellular G1 and S-Phase) Assay

For determination of cellular fractions in various stages of the cellcycle following various treatments, HS68 cells (human skin fibroblastcell line (Rb-positive)) are stained with propidium iodide stainingsolution and run on Dako Cyan Flow Cytometer. The fraction of cells inG0-G1 DNA cell cycle versus the fraction in S-phase DNA cell cycle isdetermined using FlowJo 7.2.2 analysis.

Example 18 Cell Cycle Arrest by Tricyclic Lactams in CDK4/6-DependentCells

To test the ability of tricyclic lactams to induce a clean G1-arrest, acell based screening method is used consisting of two CDK4/6-dependentcell lines (tHS68 and WM2664; Rb-positive) and one CDK4/6-independent(A2058; Rb-negative) cell line. Twenty-four hours after plating, eachcell line is treated with a tricyclic lactam compound in a dosedependent manner for 24 hours. At the conclusion of the experiment,cells are harvested, fixed, and stained with propidium iodide (a DNAintercalator), which fluoresces strongly red (emission maximum 637 nm)when excited by 488 nm light. Samples are run on Dako Cyan flowcytometer and >10,000 events are collected for each sample. Data areanalyzed using FlowJo 2.2 software developed by TreeStar, Inc.

Example 19 Inhibition of RB Phosphorylation

The CDK4/6-cyclin D complex is essential for progression from G1 to theS-phase of the DNA cell cycle. This complex phosphorylates theretinoblastoma tumor suppressor protein (Rb). To demonstrate the impactof CDK4/6 inhibition on Rb phosphorylation (pRb), tricyclic lactamcompounds are exposed to three cell lines, two CDK4/6 dependent (tHS68,WM2664; Rb-positive) and one CDK4/6 independent (A2058; Rb-negative).Twenty four hours after seeding, cells are treated with a tricycliclactam compound at 300 nM final concentration for 4, 8, 16, and 24hours. Samples are lysed and protein is assayed by western blotanalysis. Rb phosphorylation is measured at two sites targeted by theCDK4/6-cyclin D complex, Ser780 and Ser807/811 using species specificantibodies.

Example 20 Growth Arrest of Small Cell Lung Cancer (SCLC) Cells

The retinoblastoma (RB) tumor suppressor is a major negative cell cycleregulator that is inactivated in approximately 11% of all human cancers.Functional loss of RB is an obligate event in small cell lung cancer(SCLC) development. In RB competent tumors, activated CDK2/4/6 promoteG1 to S phase traversal by phosphorylating and inactivating RB (andrelated family members). Conversely, cancers with RB deletion orinactivation do not require CDK4/6 activity for cell cycle progression.

Tricyclic lactam compounds are tested for their ability to block cellproliferation in a panel of SCLC cell lines with known genetic loss ofRB. SCLC cells are treated with DMSO or a tricyclic lactam for 24 hours.The effect of tricyclic lactam compounds on proliferation is measured byEdU incorporation. An RB-intact, CDK4/6-dependent cell line (WM2664 ortHS68) and a panel of RB-negative SCLC cell lines (H69, H82, H209, H345,NCI417, or SHP-77) are analyzed for growth inhibition by the varioustricyclic lactams.

Example 21 Growth Arrest of Rb-Negative Cancer Cells

Cellular proliferation assays are conducted using the followingRb-negative cancer cell lines: H69 (human small cell lungcancer—Rb-negative) cells or A2058 (human metastatic melanomacells—Rb-negative). These cells are seeded in Costar (Tewksbury, Mass.)3093 96 well tissue culture treated white walled/clear bottom plates.Cells are treated with the tricyclic lactam compounds as nine point doseresponse dilution series from 10 uM to 1 nM. Cells are exposed tocompounds and then cell viability is determined after either four (H69)or six (A2058) days using the CellTiter-Glo® luminescent cell viabilityassay (CTG; Promega, Madison, Wis., United States of America) followingthe manufacturer's recommendations. Plates are read on a BioTek(Winooski, Vt.) Syngergy2 multi-mode plate reader. The Relative LightUnits (RLU) are plotted as a result of variable molar concentration anddata is analyzed using Graphpad (LaJolla, Californaia) Prism 5statistical software to determine the EC₅₀ for each compound.

Example 22 Bone Marrow Proliferation as Evaluated Using EdUIncorporation and Flow Cytometry Analysis

For hematopoietic stem cell and/or hematopoietic progenitor cell (HSPC)proliferation proliferation experiments, young adult female FVB/N miceare treated with a single dose of the tricyclic lactams by oral gavage.Mice are then sacrificed at the indicated times (0, 12, 24, 36, or 48hours following compound administration), and bone marrow is harvested,as previously described (Johnson et al. J. Clin. Invest. (2010) 120(7),2528-2536). Four hours before the bone marrow is harvested, mice aretreated with 100 μg of EdU by intraperitoneal injection (Invitrogen).Bone marrow mononuclear cells are harvested and immunophenotyped usingpreviously described methods and percent EdU positive cells are thendetermined (Johnson et al. J. Clin. Invest. (2010) 120(7), 2528-2536).In brief, HSPCs are identified by expression of lineage markers (Lin−),Scal (S+), and c-Kit (K+). P

Example 23 Cellular Wash-Out Experiment

HS68 cells are seeded out at 40,000 cells/well in a 60 mm dish on day 1in DMEM containing 10% fetal bovine serum, 100 U/mlpenicillin/streptomycin and 1× Glutamax (Invitrogen) as described(Brookes et al. EMBO J, 21(12) 2936-2945 (2002) and Ruas et al. Mol CellBiol, 27(12) 4273-4282 (2007)). 24 hrs post seeding, cells are treatedwith a tricyclic lactam compound or DMSO vehicle alone at 300 nM finalconcentration of test compounds. On day 3, one set of treated cellsamples are harvested in triplicate (0 Hour sample). Remaining cells arewashed two times in PBS-CMF and returned to culture media lacking testcompound. Sets of samples are harvested in triplicate at 24, 40, and 48hours.

Alternatively, the same experiment is done using normal Renal ProximalTubule Epithelial Cells (Rb-positive) obtained from American TypeCulture Collection (ATCC, Manassas, Va.). Cells are grown in anincubator at 37° C. in a humidified atmosphere of 5% CO₂ in RenalEpithelial Cell Basal Media (ATCC) supplemented with Renal EpithelialCell Growth Kit (ATCC) in 37° C. humidified incubator.

Upon harvesting cells, samples are stained with propidium iodidestaining solution and samples run on Dako Cyan Flow Cytometer. Thefraction of cells in G0-G1 DNA cell cycle versus the fraction in S-phaseDNA cell cycle is determined using FlowJo 7.2.2 analysis.

Example 24 Pharmacokinetic and Pharmacodynamic Properties of TricyclicLactams

Tricyclic lactam compounds can be dosed to mice at 30 mg/kg by oralgavage or 10 mg/kg by intravenous injection. Blood samples are taken at0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 hours post dosing and the plasmaconcentrations of the tricyclic lactam compounds are determined by HPLC.

Example 25 Metabolic Stability

The metabolic stability of tricyclic lactam compounds can be determinedin human, dog, rat, monkey, and mouse liver microsomes. Human, mouse,and dog liver microsomes are purchased from Xenotech, and Sprague-Dawleyrat liver microsomes are prepared by Absorption Systems. The reactionmixture comprising 0.5 mg/mL of liver microsomes, 100 mM of potassiumphosphate, pH 7.4, 5 mM of magnesium chloride, and 1 uM of test compoundis prepared. The test compound is added into the reaction mixture at afinal concentration of 1 uM. An aliquot of the reaction mixture (withoutcofactor) is incubated in shaking water bath at 37° C. for 3 minutes.The control compound, testosterone, is run simultaneously with the testcompound in a separate reaction. The reaction is initiated by theaddition of cofactor (NADPH), and the mixture is then incubated in ashaking water bath at 37° C. Aliquots (100 μL) are withdrawn at 0, 10,20, 30, and 60 minutes for the test compound and 0, 10, 30, and 60minutes for testosterone. Test compound samples are immediately combinedwith 100 μL of ice-cold acetonitrile containing internal standard toterminate the reaction. Testosterone samples are immediately combinedwith 800 μL of ice cold 50/50 acetonitrile/dH₂O containing 0.1% formicacid and internal standard to terminate the reaction. The samples areassayed using a validated LC-MS/MS method. Test compound samples areanalyzed using the Orbitrap high resolution mass spectrometer toquantify the disappearance of parent test compound and detect theappearance of metabolites. The peak area response ration (PARR) tointernal standard is compared to the PARR at time 0 to determine thepercent of test compound or positive control remaining at time-point.Half-lives are calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Half-life is calculated basedon t½=0.693k, where k is the elimination rate constant based on theslope plot of natural logarithm percent remaining versus incubationtime.

Example 26 Resistance to Chemotherapy-Induced Cell Death, DNA Damage,and Caspase Activation

In order to demonstrate that pharmacological quiescence induced bytricyclic lactam compound treatment affords resistance tochemotherapeutic agents with differing mechanisms of action, an in vitromodel is developed using telomerized human diploid fibroblasts (tHDFs; ahuman foreskin fibroblast line immortalized with expression of humantelomerase). These cells are highly CDK4/6-dependent for proliferationas demonstrated by their complete G1 arrest following treatment withCDK4/6 inhibitors (See Roberts P J, et al. Multiple Roles ofCyclin-Dependent Kinase 4/6 Inhibitors in Cancer Therapy. J Natl CancerInst 2012; Mar. 21; 104(6): 476-87). Cell survival is determined by CellTiterGlo assay per manufacturer's recommendations. For both γ-H2AX andcaspase 3/7 assays, cells are plated and allowed to become adherent for24 hours. Cells are then treated with tricyclic lactam compounds (atindicated concentrations) or vehicle control for 16 hours, at which timethe indicated chemotherapy is added to the pretreated cells. For γ-H2AX,cells are harvested for analysis 8 hours after chemotherapy exposure.For the γ-H2AX assay, cells are fixed, permeabilized, and stained withanti-γ-H2AX as per the γ-H2AX Flow Kit (Millipore) and quantitated byflow cytometry. Data is analyzed using FlowJo 2.2 software developed byTreeStar, Inc. For the in vitro caspase 3/7 assay, cells are harvested24 hours post chemotherapy treatment. Caspase 3/7 activation is measuredusing the Caspase-Glo® 3/7 Assay System (Promega) per manufacturer'srecommendations.

Example 27 Inhibition of Hematopoietic Stem and/or Progenitor Cell(HSPC) Proliferation

To characterize the effect of tricyclic lactam compound treatment onproliferation of the different mouse hematopoietic cells, 8-week-oldfemale C57Bl/6 mice are given a single dose of vehicle alone (20%Solutol) or a tricyclic lactam compound (150 mg/kg) by oral gavage.Ten-hours later, all mice are given a single i.p. injection of 100 mcgEdU (5-ethynyl-2′-deoxyuridine) to label cells in S-phase of the cellcycle. All treated mice are euthanized 2 hours after EdU injection, bonemarrow cells are harvested and processed for flow cytometric analysis ofEdU-incorporation.

Example 28 Inhibition of Differentiated Hematopoietic Cell Proliferation

Using the same experimental protocol as discussed in Example 27, theeffect of tricyclic lactam compounds on the proliferation ofdifferentiated hematopoietic cells is investigated.

Example 29 Protection of Bone Marrow Progenitors by Tricyclic LactamCompounds

To assess the effect of tricyclic lactam compounds oncarboplatin-induced cytotoxicity in the bone marrow, FVB/n mice aretreated with vehicle control, 90 mg/kg carboplatin by intraperitonealinjection, or 150 mg/kg tricyclic lactam compound by oral gavage plus 90mg/kg carboplatin by intraperitoneal injection. Twenty four hours aftertreatment, bone marrow is harvested and the percent of cycling bonemarrow progenitors is measured by EdU incorporation.

Example 30 Effect of Tricyclic Lactam Compounds on 5FU-InducedMyelosuppression

To determine the ability of tricyclic lactam compounds to modulatechemotherapy-induced myelosuppression, a well characterized single-dose5-fluorouracil (5FU) regimen, known to be highly myelosuppressive inmice, is utilized. FVB/n female mice are given single oral doses ofvehicle or tricyclic lactam compound at 150 mg/kg, followed 30 minuteslater by a single intraperitoneal dose of 5FU at 150 mg/kg. Completeblood cell counts are measured every two days starting on day six.

Example 31 Effect of Tricyclic Lactam Compounds on 5FU-InducedMyelosuppression Through Repeated Cycles of 5FU Treatment

To determine the ability of tricyclic lactam compounds to modulatechemotherapy-induced myelosuppression through repeated cycles ofchemotherapy, a well characterized 5-fluorouracil (5FU) regimen, knownto be highly myelosuppressive in mice is utilized. 8-week-old femaleC57Bl/6 mice are given a single oral dose of vehicle (20% Solutol) ortricyclic lactam compound at 150 mg/kg followed 30 minutes later by anintraperitoneal dose of 5FU at 150 mg/kg. This is repeated every 21 daysfor 3 cycles. Blood samples are taken for hematology analysis on Day 10of Cycles 1-3.

Example 32 DNA Cell Cycle Analysis in Human Renal Proximal Tubule Cells

To test the ability of tricyclic lactams to induce a clean G1-arrest innon-hematopoietic cells, G1 arrest is examined in human renal proximaltubule cells. The cells are treated with tricyclic lactam compounds in adose dependent manner for 24 hours. At the conclusion of the experiment,cells are harvested, fixed, and stained with propidium iodide (a DNAintercalator), which fluoresces strongly red (emission maximum 637 nm)when excited by 488 nm light. Samples are run on a Dako Cyan flowcytometer. Data are analyzed using FlowJo 2.2 software developed byTreeStar, Inc. Assays are run in triplicate.

Example 33 Protection of Renal Proximal Tubule Epithelial Cells fromChemotherapy-Induced DNA Damage by Tricyclic Lactam Compounds

The ability of tricyclic lactams to protect human renal proximal tubulecells from chemotherapy induced DNA damage is analyzed using etoposideand cisplatin. The cells are treated with a tricyclic lactam compound ina dose dependent manner (10 nM, 30 nM, 100 nM, 300 nM, or 1000 nM). Atthe conclusion of the experiment, cells are harvested, fixed, andstained with propidium iodide (a DNA intercalator), which fluorescesstrongly red (emission maximum 637 nm) when excited by 488 nm light.Samples are run on Dako Cyan flow cytometer. Data are analyzed usingFlowJo 2.2 software developed by TreeStar, Inc.

Example 34 Prevention of Chemotherapy-Induced DNA Damage and CaspaseActivation in Human Renal Proximal Tubule Cells by Tricyclic LactamCompounds

In order to demonstrate that pharmacological quiescence induced bytricyclic lactam treatment affords resistance to chemotherapeutic agentsin non-hematopoietic cells, the protective effect of tricyclic lactamcompounds on human renal proximal tubule cells is analyzed. Normal renalproximal tubule epithelial cells are obtained from American Type CultureCollection (ATCC, Manassas, Va.). Cells are grown in an incubator at 37°C. in a humidified atmosphere of 5% CO₂ in Renal Epithelial Cell BasalMedia (ATCC) supplemented with a Renal Epithelial Cell Growth Kit (ATCC)in a 37° C. humidified incubator. Cells are treated with either DMSO or10 nM, 30 nM, 100 nM, 300 nM or 1 uM tricyclic lactam compound in eitherthe absence or presence of 25 uM cisplatin. For the γ-H2AX assay, cellsare fixed, permeabilized, and stained with anti-γ-H2AX as per the γ-H2AXFlow Kit (Millipore) and quantitated by flow cytometry. Data is analyzedusing FlowJo 2.2 software developed by TreeStar, Inc. Caspase 3/7activation is measured using the Caspase-Glo 3/7 Assay System (Promega,Madison, Wis.) by following the manufacturer's instructions.

Example 35 Preparation of Drug Product

The active compounds of the present invention can be prepared forintravenous administration using the following procedure. The excipientshydroxypropyl-beta-cyclodextrin and dextrose can be added to 90% of thebatch volume of USP Sterile Water for Injection or Irrigation withstirring; stir until dissolved. The active compound in the hydrochloridesalt form is added and stirred until it is dissolved. The pH is adjustedwith 1N NaOH to pH 4.3+0.1 and 1N HCl can be used to back titrate ifnecessary. USP sterile water for injection or irrigation can be used tobring the solution to the final batch weight. The pH is next re-checkedto ensure that the pH is pH 4.3+0.1. If the pH is outside of the rangeadd 1N HCl or 1N NaOH as appropriate to bring the pH to 4.3+0.1. Thesolution is next sterile filtered to fill 50 or 100 mL flint glassvials, stopper, and crimped.

This specification has been described with reference to embodiments ofthe invention. The invention has been described with reference toassorted embodiments, which are illustrated by the accompanyingExamples. The invention can, however, be embodied in different forms andshould not be construed as limited to the embodiments set forth herein.Given the teaching herein, one of ordinary skill in the art will be ableto modify the invention for a desired purpose and such variations areconsidered within the scope of the invention.

We claim:
 1. A method of reducing the effect of chemotherapy on healthycells in a subject being treated for cyclin-dependent kinase 4/6(CDK4/6) replication independent cancer or abnormal cell proliferation,wherein said healthy cells are hematopoietic stem cells, hematopoieticprogenitor cells, or renal epithelial cells, the method comprisingadministering to the subject, an effective amount of an compoundselected from the group consisting of Formula I, II, III, IV, or V:

wherein: Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)—wherein z is 2, 3 or 4; each X is independently CH or N; each X′ isindependently CH or N; X″ is independently CH₂, S or NH, arranged suchthat the moiety is a stable 5-membered ring; R, R⁸, and R¹¹ areindependently H, C₁-C₃ alkyl or haloalkyl, cycloalkyl or cycloalkylcontaining one or more heteroatoms selected from N, O or S;-(alkylene)_(m)-C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl,-(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)n-NR³R⁴ any of whichmay be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atoms may optionally combine to form a ring; each R¹ isindependently aryl, alkyl, cycloalkyl or haloalkyl, wherein each of saidalkyl, cycloalkyl and haloalkyl groups optionally includes O or Nheteroatoms in place of a carbon in the chain and two R¹'s on adjacentring atoms or on the same ring atom together with the ring atom(s) towhich they are attached optionally form a 3-8-membered cycle; y is 0, 1,2, 3 or 4; R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-C(O)—O-alkyl;-(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or-(alkylene)_(m)-S(O)_(n)—NR³R⁴ any of which may be optionallyindependently substituted with one or more R^(x) groups as allowed byvalance, and wherein two R^(x) groups bound to the same or adjacent atommay optionally combine to form a ring and wherein m is 0, 1 or 2 and nis 0, 1 or 2; R³ and R⁴ at each occurrence are independently: (i)hydrogen or (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring; or R³ andR⁴ together with the nitrogen atom to which they are attached maycombine to form a heterocyclo ring optionally independently substitutedwith one or more R^(x) groups as allowed by valance, and wherein twoR^(x) groups bound to the same or adjacent atom may optionally combineto form a ring; R⁵ and R⁵* at each occurrence is: (i) hydrogen or (ii)alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance; R^(x) at each occurrence is independently,halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl,cycloalkylalkyl, heterocycloalkyl, -(alkylene)_(m)-OR⁵,-(alkylene)_(m)-O-alkylene-OR⁵, -(alkylene)_(m)-S(O)_(n)—R⁵,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵,-(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵,-(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵,-(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(S)—NR³R⁴,-(alkylene)_(m)-N(R³)—C(O)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴,-(alkylene)_(m)-N(R³)—C(O)—R⁵, -(alkylene)_(m)-N(R³)—C(S)—R⁵,-(alkylene)_(m)-O—C(O)—NR³R⁴, -(alkylene)_(m)-O—C(S)—NR³R⁴,-(alkylene)_(m)-SO₂—NR³R⁴, -(alkylene)_(m)-N(R³)—SO₂—R⁵,-(alkylene)_(m)-N(R³)—SO₂—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—OR⁵)-(alkylene)_(m)-N(R³)—C(S)—OR⁵, or -(alkylene)_(m)-N(R³)—SO₂—R⁵;wherein: said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl,cycloalkylalkyl, and heterocycloalkyl groups may be furtherindependently substituted with one or more -(alkylene)_(m)-CN,-(alkylene)_(m)-OR⁵*, -(alkylene)_(m)-S(O)_(n)—R⁵*,-(alkylene)_(m)-NR³*R⁴*, -(alkylene)_(m)-C(O)—R⁵*,-(alkylene)_(m)-C(═S)R⁵*, -(alkylene)_(m)-C(═O)OR⁵*,-(alkylene)_(m)-OC(═O)R⁵*, -(alkylene)_(m)-C(S)—OR⁵*,-(alkylene)_(m)-C(O)—NR³*R⁴*, -(alkylene)_(m)-C(S)—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,-(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,-(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—SO₂—R⁵*, -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—C(O)—OR⁵*, -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or-(alkylene)_(m)-N(R³*)—SO₂—R⁵*, n is 0, 1 or 2, and m is 0, 1 or 2; R³*and R⁴* at each occurrence are independently: (i) hydrogen or (ii)alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl, heteroaryl,cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance; or R³* and R⁴* together with the nitrogenatom to which they are attached may combine to form a heterocyclo ringoptionally independently substituted with one or more R^(x) groups asallowed by valance; and R⁶ is H or lower alkyl,-(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atoms may optionally combine to form a ring; and R¹⁰ is(i) NHR^(A), wherein R^(A) is unsubstituted or substituted C₁-C₈ alkyl,cycloalkylalkyl, or -TT-RR, C₁-C₈ cycloalkyl or cycloalkyl containingone or more heteroatoms selected from N, O, and S; TT is anunsubstituted or substituted C₁-C₈ alkyl or C₃-C₈ cycloalkyl linker; andRR is a hydroxyl, unsubstituted or substituted C₁-C₆ alkoxy, amino,unsubstituted or substituted C₁-C₆ alkylamino, unsubstituted orsubstituted di-C₁-C₆ alkylamino, unsubstituted or substituted C₆-C₁₀aryl, unsubstituted or substituted heteroaryl comprising one or two 5-or 6-member rings and 1-4 heteroatoms selected from N, O and S,unsubstituted or substituted C₃-C₁₀ carbocycle, or unsubstituted orsubstituted heterocycle comprising one or two 5- or 6-member rings and1-4 heteroatoms selected from N, O and S; or (ii) —C(O)—R¹² or—C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³ is R^(A); whencompounds comprise a double bond in the 6-membered ring fused to thepyrimidine ring, two R⁸ groups are present and are as defined above;when compounds do not comprise a double bond in the 6-membered ringfused to the pyrimidine ring, four R⁸ groups are present and are asdefined above; or a pharmaceutically acceptable salt thereof.
 2. Themethod of claim 1, wherein each R⁸ is independently hydrogen or C₁-C₃alkyl.
 3. The method of claim 1, wherein the compound has the formulaselected from the structures shown in FIG.
 5. 4. The method of claim 1,wherein the compound has the formula selected from the structures shownin FIG.
 6. 5. The method of claim 1, wherein the compound has theformula selected from the structures shown in FIG.
 7. 6. The method ofclaim 1, wherein the compound has the formula selected from thestructures shown in FIG.
 8. 7. The method of claim 1, wherein thecompound has the formula selected from the structures shown in FIG. 9.8. The method of claim 1, wherein the compound has the formula:


9. The method of claim 1, wherein the compound has the formula:


10. The method of claim 1, wherein the compound has the formula:


11. The method of claim 1, wherein the compound has the formula:


12. The method of claim 1, wherein the compound has the formula:


13. The method of claim 1, wherein the compound has the formula:


14. The method of claim 1, wherein the compound has the formula:


15. The method of claim 1, wherein the compound has the formula:


16. The method of claim 1, wherein the compound has the formula:


17. The method of claim 1, wherein the compound has the formula:


18. The method of claim 1, wherein the compound has the formula:


19. The method of claim 1, wherein the compound has the formula:


20. The method of claim 1, wherein the compound has the formula:


21. The method of claim 1, wherein the compound has the formula:


22. The method of claim 1, wherein the compound has the formula:


23. The method of claim 1, wherein the compound has the formula:


24. The method of claim 1, wherein the compound is selected from thegroup consisting of: Structure Reference Structure A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

BB

CC

DD

EE

FF

GG

HH

II

JJ

KK

LL

MM

NN

OO

PP

QQ

RR

SS

TT

UU

VV

WW

XX

YY

ZZ

AAA

BBB

CCC

DDD

EEE

FFF

GGG

HHH

III

JJJ

KKK

LLL

MMM

NNN

OOO

PPP

QQQ

RRR

SSS

TTT

UUU

VVV

WWW

XXX


25. The method of claim 1, wherein the subject is a human.
 26. Themethod of claim 1, wherein the compound is administered to the subject24 hours or less prior to exposure to the cytotoxic compound.
 27. Themethod of claim 1, wherein the subject has cancer.
 28. The method ofclaim 1, wherein the subject has an abnormal cell proliferation.
 29. Themethod of claim 1, wherein the cancer or abnormal cell proliferation ischaracterized by a loss or absence of retinoblastoma tumor suppressorprotein (RB).
 30. The method of claim 1, wherein the cancer is smallcell lung cancer, retinoblastoma, triple negative breast cancer, humanpapillomavirus (HPV) positive head and neck cancer, or HPV positivecervical cancer.
 31. The method of claim 1, wherein the chemotherapy isselected from an alkylating agent, DNA intercalator, protein synthesisinhibitor, inhibitor of DNA or RNA synthesis, DNA base analog,topoisomerase inhibitor, telomerase inhibitor or telomeric DNA bindingcompound.
 32. The method of claim 1, wherein the cancer is small celllung carcinoma and the chemotherapy is selected from the groupconsisting of etoposide, cisplatin, and carboplatin, or a combinationthereof.
 33. The method of claim 1, wherein at least 80% or more of theHSPCs re-enter the cell cycle in less than 36 hours from the lastadministration of the compound.
 34. The method of claim 1, wherein thehealthy cells are hematopoietic stem cells or hematopoietic progenitorcells.
 35. The method of claim 1, wherein the healthy cells are renalepithelial cells.
 36. A method of treating an Rb-negative cancer orabnormal cell proliferation in a subject with combination therapy,comprising administering to the subject an effective amount of acombination of chemotherapy and a compound selected from the groupconsisting of Formula I, II, III, IV, or V:

wherein: Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)—wherein z is 2, 3 or 4; each X is independently CH or N; each X′ isindependently CH or N; X″ is independently CH₂, S or NH, arranged suchthat the moiety is a stable 5-membered ring; R, R⁸, and R¹¹ areindependently H, C₁-C₃ alkyl or haloalkyl, cycloalkyl or cycloalkylcontaining one or more heteroatoms selected from N, O or S;-(alkylene)_(m)-C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl,-(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atoms may optionally combine to form a ring; each R¹ isindependently aryl, alkyl, cycloalkyl or haloalkyl, wherein each of saidalkyl, cycloalkyl and haloalkyl groups optionally includes O or Nheteroatoms in place of a carbon in the chain and two R¹'s on adjacentring atoms or on the same ring atom together with the ring atom(s) towhich they are attached optionally form a 3-8-membered cycle; y is 0, 1,2, 3 or 4; R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-C(O)—O-alkyl;-(alkylene)_(m)-O—R⁵, -(alkylene)_(m)-S(O)_(n)—R⁵, or-(alkylene)_(m)-S(O)_(n)—NR³R⁴ any of which may be optionallyindependently substituted with one or more R^(x) groups as allowed byvalance, and wherein two R^(x) groups bound to the same or adjacent atommay optionally combine to form a ring and wherein m is 0, 1 or 2 and nis 0, 1 or 2; R³ and R⁴ at each occurrence are independently: (i)hydrogen or (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring; or R³ andR⁴ together with the nitrogen atom to which they are attached maycombine to form a heterocyclo ring optionally independently substitutedwith one or more R^(x) groups as allowed by valance, and wherein twoR^(x) groups bound to the same or adjacent atom may optionally combineto form a ring; R⁵ and R⁵* at each occurrence is: (i) hydrogen or (ii)alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance; R^(x) at each occurrence is independently,halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl,cycloalkylalkyl, heterocycloalkyl, -(alkylene)_(m)-OR⁵,-(alkylene)_(m)-O-alkylene-OR⁵, -(alkylene)_(m)-S(O)_(n)—R⁵,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵,-(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵,-(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵,-(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(S)—NR³R⁴,-(alkylene)_(m)-N(R³)—C(O)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴,-(alkylene)_(m)-N(R³)—C(O)—R⁵, -(alkylene)_(m)-N(R³)—C(S)—R⁵,-(alkylene)_(m)-O—C(O)—NR³R⁴, -(alkylene)_(m)-O—C(S)—NR³R⁴,-(alkylene)_(m)-SO₂—NR³R⁴, -(alkylene)_(m)-N(R³)—SO₂—R⁵,-(alkylene)_(m)-N(R³)—SO₂—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—OR⁵)-(alkylene)_(m)-N(R³)—C(S)—OR⁵, or -(alkylene)_(m)-N(R³)—SO₂—R⁵;wherein: said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl, heteroarylalkyl,cycloalkylalkyl, and heterocycloalkyl groups may be furtherindependently substituted with one or more -(alkylene)_(m)-CN,-(alkylene)_(m)-OR⁵*, -(alkylene)_(m)-S(O)_(n)—R⁵*,-(alkylene)_(m)-NR³*R⁴*, -(alkylene)_(m)-C(O)—R⁵*,-(alkylene)_(m)-C(═S)R⁵*, -(alkylene)_(m)-C(═O)OR⁵*,-(alkylene)_(m)-OC(═O)R⁵*, -(alkylene)_(m)-C(S)—OR⁵*,-(alkylene)_(m)-C(O)—NR³*R⁴*, -(alkylene)_(m)-C(S)—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,-(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,-(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—SO₂—R⁵*, -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—C(O)—OR⁵*, -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or-(alkylene)_(m)-N(R³*)—SO₂—R⁵*, n is 0, 1 or 2, and m is 0, 1 or 2; R³*and R⁴* at each occurrence are independently: (i) hydrogen or (ii)alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl, heteroaryl,cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance; or R³* and R⁴* together with the nitrogenatom to which they are attached may combine to form a heterocyclo ringoptionally independently substituted with one or more R^(x) groups asallowed by valance; and R⁶ is H or lower alkyl,-(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atoms may optionally combine to form a ring; and R¹⁰ is1 (i) NHR^(A), wherein R^(A) is unsubstituted or substituted C₁-C₈alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈ cycloalkyl or cycloalkylcontaining one or more heteroatoms selected from N, O, and S; TT is anunsubstituted or substituted C₁-C₈ alkyl or C₃-C₈ cycloalkyl linker; andRR is a hydroxyl, unsubstituted or substituted C₁-C₆ alkoxy, amino,unsubstituted or substituted C₁-C₆ alkylamino, unsubstituted orsubstituted di-C₁-C₆ alkylamino, unsubstituted or substituted C₆-C₁₀aryl, unsubstituted or substituted heteroaryl comprising one or two 5-or 6-member rings and 1-4 heteroatoms selected from N, O and S,unsubstituted or substituted C₃-C₁₀ carbocycle, or unsubstituted orsubstituted heterocycle comprising one or two 5- or 6-member rings and1-4 heteroatoms selected from N, O and S; or (ii) —C(O)—R¹² or—C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³ is R^(A); whencompounds comprise a double bond in the 6-membered ring fused to thepyrimidine ring, two R⁸ groups are present and are as defined above;when compounds do not comprise a double bond in the 6-membered ringfused to the pyrimidine ring, four R⁸ groups are present and are asdefined above; or a pharmaceutically acceptable salt thereof.
 37. Themethod of claim 36, wherein the compound is selected from the groupconsisting of: Structure Reference Structure A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

BB

CC

DD

EE

FF

GG

HH

II

JJ

KK

LL

MM

NN

OO

PP

QQ

RR

SS

TT

UU

VV

WW

XX

YY

ZZ

AAA

BBB

CCC

DDD

EEE

FFF

GGG

HHH

III

JJJ

KKK

LLL

MMM

NNN

OOO

PPP

QQQ

RRR

SSS

TTT

UUU

VVV

WWW

XXX


38. A method of reducing the effect of chemotherapy on healthy cells ina subject being treated for cyclin-dependent kinase 4/6 (CDK4/6)replication independent cancer or abnormal cell proliferation, whereinsaid healthy cells are hematopoietic stem cells, hematopoieticprogenitor cells, or renal epithelial cells, the method comprisingadministering to the subject, an effective amount of an compoundselected from the group consisting of Formula VI:

wherein R, R¹, R², R³, R⁴, R⁵, R⁶, R^(x), Z, m, n, and y are as definedin claim 1; each R¹⁴ is independently H, C₁-C₃ alkyl (including methyl)or haloalkyl, cycloalkyl or cycloalkyl containing one or moreheteroatoms selected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl,-(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valence, and wherein two R^(x) groups bound to thesame or adjacent atoms may optionally combine to form a ring; or two R¹⁴groups bonded to the same carbon can form an exocyclic double bond; ortwo R¹⁴ groups bonded to the same carbon can form a carbonyl group; andwhen the compound of Formula VI has a double bond, as indicated by the( - - - - ), in the 6-membered ring fused to the pyrimidine ring, twoR¹⁴ groups are present as allowed for in Formula VI above; or when thecompound of Formula VI does not include a double bond, as indicated bythe ( - - - - ), in the 6-membered ring fused to the pyrimidine ring,four R¹⁴ groups are present as allowed for in Formula VI above; or apharmaceutically acceptable salt thereof.
 39. The method of claim 1,wherein the compound is selected from the group consisting of: XXX

ZZZ

BBBB

DDDD

EEEE

or a pharmaceutically acceptable salt thereof.
 40. The method of claim38, wherein the compound is selected from the group consisting of: YYY

AAAA

CCCC

FFFF

GGGG

HHHH

or a pharmaceutically acceptable salt thereof.
 41. A method of reducingthe effect of chemotherapy on healthy cells in a subject being treatedfor cyclin-dependent kinase 4/6 (CDK4/6) replication independent canceror abnormal cell proliferation, wherein said healthy cells arehematopoietic stem cells, hematopoietic progenitor cells, or renalepithelial cells, the method comprising administering to the subject, aneffective amount of an compound selected from the group consisting ofFormula I, II, III, IV, or V:

wherein: Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)—wherein z is 2, 3 or 4; each X is independently CH or N; each X′ isindependently CH or N; X″ is independently CH₂, S or NH, arranged suchthat the moiety is a stable 5-membered ring; R, R⁸, and R¹¹ areindependently H, C₁-C₃ alkyl (including methyl) or haloalkyl, cycloalkylor cycloalkyl containing one or more heteroatoms selected from N, O orS; -(alkylene)_(m)-C₃-C₈ cycloalkyl, -(alkylene)_(m)-aryl,-(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich, other than heterocyclo, may be optionally independentlysubstituted with one or more R^(x) groups as allowed by valence, andwherein two R^(x) groups bound to the same or adjacent atoms mayoptionally combine to form a ring; each R¹ is independently aryl, alkyl,cycloalkyl or haloalkyl, wherein each of said alkyl, cycloalkyl andhaloalkyl groups optionally includes O or N heteroatoms in place of acarbon in the chain and two R¹'s on adjacent ring atoms or on the samering atom together with the ring atom(s) to which they are attachedoptionally form a 3-8-membered cycle; y is 0, 1, 2, 3 or 4; R² is-(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴;-(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich, other than heterocyclo, may be optionally independentlysubstituted with one or more R^(x) groups as allowed by valance, andwherein two R^(x) groups bound to the same or adjacent atom mayoptionally combine to form a ring and wherein m is 0, 1, or 2 and n is0, 1 or 2; wherein heterocyclo may be optionally independentlysubstituted with 1 to 3 R^(x) groups as allowed by valance, and whereintwo R^(x) groups bound to the same or adjacent atom may optionallycombine to form a ring; R³ and R⁴ at each occurrence are independently:(i) hydrogen or (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any ofwhich, other than heterocyclo, may be optionally independentlysubstituted with one or more R^(x) groups as allowed by valance, andwherein two R^(x) groups bound to the same or adjacent atom mayoptionally combine to form a ring; or R³ and R⁴ together with thenitrogen atom to which they are attached may combine to form aheterocyclo ring optionally independently substituted with one or moreR^(x) groups as allowed by valance, and wherein two R^(x) groups boundto the same or adjacent atom may optionally combine to form a ring; R⁵and R⁵* at each occurrence is: (i) hydrogen or (ii) alkyl, alkenyl,alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkylalkyl,heterocycloalkyl, arylalkyl, or heteroarylalkyl any of which, other thanheterocyclo, may be optionally independently substituted with one ormore R^(x) groups as allowed by valance; R^(x) at each occurrence isindependently, halo, cyano, nitro, oxo, alkyl, haloalkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl,arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl,-(alkylene)_(m)-OR⁵, -(alkylene)_(m)-O-alkylene-OR⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-CN,-(alkylene)_(m)-C(O)—R⁵, -(alkylene)_(m)-C(S)—R⁵,-(alkylene)_(m)-C(O)—OR⁵, -(alkylene)_(m)-O—C(O)—R⁵,-(alkylene)_(m)-C(S)—OR⁵, -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴,-(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵,-(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴,-(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-SO₂—NR³R⁴,-(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴,-(alkylene)_(m)-N(R³)—C(O)—OR⁵, -(alkylene)_(m)-N(R³)—C(S)—OR⁵, or-(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein: said alkyl, haloalkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, heterocyclo, aryl, heteroaryl,arylalkyl, heteroarylalkyl, cycloalkylalkyl, and heterocycloalkylgroups, any of which, other than heterocyclo, may be furtherindependently substituted with one or more -(alkylene)_(m)-CN,-(alkylene)_(m)-OR⁵*, -(alkylene)_(m)-S(O)_(n)—R⁵*,-(alkylene)_(m)-NR³*R⁴*, -(alkylene)_(m)-C(O)—R⁵*,-(alkylene)_(m)-C(═S)R⁵*, -(alkylene)_(m)-C(═O)OR⁵*,-(alkylene)_(m)-OC(═O)R⁵*, -(alkylene)_(m)-C(S)—OR⁵*,-(alkylene)_(m)-C(O)—NR³*R⁴*, -(alkylene)_(m)-C(S)—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,-(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,-(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—SO₂—R⁵*, -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—C(O)—OR⁵*, -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or-(alkylene)_(m)-N(R³*)—SO₂—R⁵*, and wherein heterocycle may be furtherindependently substituted with one to three substitutions selected from-(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵*, -(alkylene)_(m)-S(O)_(n)—R⁵*,-(alkylene)_(m)-NR³*R⁴*, -(alkylene)_(m)-C(O)—R⁵*,-(alkylene)_(m)-C(═S)R⁵*, -(alkylene)_(m)-C(═O)OR⁵*,-(alkylene)_(m)-OC(═O)R⁵*, -(alkylene)_(m)-C(S)—OR⁵*,-(alkylene)_(m)-C(O)—NR³*R⁴*, -(alkylene)_(m)-C(S)—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*, -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,-(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,-(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—SO₂—R⁵*, -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,-(alkylene)_(m)-N(R³*)—C(O)—OR⁵*, -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or-(alkylene)_(m)-N(R³*)—SO₂—R⁵*; n is 0, 1 or 2, and m is 0, 1; or 2 andR³* and R⁴* at each occurrence are independently: (i) hydrogen or (ii)alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl any ofwhich, other than heterocyclo, may be optionally independentlysubstituted with one or more R^(x) groups as allowed by valance; or R³*and R⁴* together with the nitrogen atom to which they are attached maycombine to form a heterocyclo ring optionally independently substitutedwith one or more R^(x) groups as allowed by valance; R⁶ is H, absent, orlower alkyl, -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich, other than heterocyclo, may be optionally independentlysubstituted with one or more R^(x) groups as allowed by valence, andwherein two R^(x) groups bound to the same or adjacent atoms mayoptionally combine to form a ring; and R¹⁰ is (i) NHR^(A), wherein R^(A)is unsubstituted or substituted C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR,C₁-C₈ cycloalkyl or cycloalkyl containing one or more heteroatomsselected from N, O, and S; TT is an unsubstituted or substituted C₁-C₈alkyl or C₃-C₈ cycloalkyl linker; and RR is a hydroxyl, unsubstituted orsubstituted C₁-C₆ alkoxy, amino, unsubstituted or substituted C₁-C₆alkylamino, unsubstituted or substituted di-C₁-C₆ alkylamino,unsubstituted or substituted C₆-C₁₀ aryl, unsubstituted or substitutedheteroaryl comprising one or two 5- or 6-member rings and 1-4heteroatoms selected from N, O and S, unsubstituted or substitutedC₃-C₁₀ carbocycle, or unsubstituted or substituted heterocyclecomprising one or two 5- or 6-member rings and 1-4 heteroatoms selectedfrom N, O and S; or (ii) —C(O)—R¹² or —C(O)O—R¹³, wherein R¹² is NHR^(A)or R^(A) and R¹³ is R^(A); when the compound of Formula I, II, III, IV,or V has a double bond, as indicated by the ( - - - - ), in the6-membered ring fused to the pyrimidine ring, two R⁸ groups are presentas allowed for in Formula I, II, III, IV, or V above; or when thecompound of Formula I, II, III, IV, or V does not include a double bond,as indicated by the ( - - - - ), in the 6-membered ring fused to thepyrimidine ring, four R⁸ groups are present as allowed for in Formula I,II, III, IV, or V above; wherein each heteroaryl is an aryl ring systemthat contains one or more heteroatoms selected from the group O, N andS, wherein the ring nitrogen and sulfur atom(s) are optionally oxidized,and nitrogen atom(s) are optionally quarternized; wherein each aryl is acarbocyclic aromatic system containing one or two rings, wherein suchrings may be attached together in a fused manner, and wherein each arylmay have 1 or more R^(x) substituents; wherein each heterocyclo is asaturated or partially saturated heteroatom-containing ring radical,where the heteroatoms may be selected from nitrogen, sulfur and oxygen,wherein each heterocyclo is a monocyclic 6-8 membered ring or a 5-16membered bicyclic ring system, and wherein each heterocyclo may have 1to 3 R^(x) substituents; or a pharmaceutically acceptable salt thereof.42. A method of reducing the effect of chemotherapy on healthy cells ina subject being treated for cyclin-dependent kinase 4/6 (CDK4/6)replication independent cancer or abnormal cell proliferation, whereinsaid healthy cells are hematopoietic stem cells, hematopoieticprogenitor cells, or renal epithelial cells, the method comprisingadministering to the subject, an effective amount of an compoundselected from the group consisting of Formula VI:

wherein R, R¹, R², R³, R⁴, R⁵, R⁶, R^(x), Z, m, n, and y are as definedin claim 41; each R¹⁴ is independently H, C₁-C₃ alkyl (including methyl)or haloalkyl, cycloalkyl or cycloalkyl containing one or moreheteroatoms selected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl,-(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich, other than heterocyclo, may be optionally independentlysubstituted with one or more R^(x) groups as allowed by valence, andwherein two R^(x) groups bound to the same or adjacent atoms mayoptionally combine to form a ring; or two R¹⁴ groups bonded to the samecarbon can form an exocyclic double bond; or two R¹⁴ groups bonded tothe same carbon can form a carbonyl group; and when the compound ofFormula VI has a double bond, as indicated by the ( - - - - ), in the6-membered ring fused to the pyrimidine ring, two R¹⁴ groups are presentas allowed for in Formula VI above; or when the compound of Formula VIdoes not include a double bond, as indicated by the ( - - - - ), in the6-membered ring fused to the pyrimidine ring, four R¹⁴ groups arepresent as allowed for in Formula VI above; wherein each heteroaryl isan aryl ring system that contains one or more heteroatoms selected fromthe group O, N and S, wherein the ring nitrogen and sulfur atom(s) areoptionally oxidized, and nitrogen atom(s) are optionally quarternized;wherein each aryl is a carbocyclic aromatic system containing one or tworings, wherein such rings may be attached together in a fused manner,and wherein each aryl may have 1 or more R^(x) substituents; whereineach heterocyclo is a saturated or partially saturatedheteroatom-containing ring radical, where the heteroatoms may beselected from nitrogen, sulfur and oxygen, wherein each heterocyclo is amonocyclic 6-8 membered ring or a 5-16 membered bicyclic ring system,and wherein each heterocyclo may have 1 to 3 R^(x) substituents; or apharmaceutically acceptable salt thereof.